Targeting agent antibody conjugates and uses thereof

ABSTRACT

Methods, compositions and uses are provided for bispecific antibodies comprising one or more unnatural amino acids. The bispecific antibodies may bind to two or more different receptors, co-receptors, antigens, or cell markers on one or more cells. The bispecific antibodies may be used to treat a disease or condition (e.g., cancer, autoimmune disease, pathogenic infection, inflammatory disease). The bispecific antibodies may be used to modulate (e.g., stimulate or suppress) an immune response.

CROSS-REFERENCE

This application is a divisional application of U.S. application Ser.No. 14/774,647 filed Sep. 10, 2015, which is a U.S. National Stage Entryof International Application No. PCT/US2014/029379 filed Mar. 14, 2014,which claims the benefit of priority from U.S. provisional applicationSer. No. 61/783,426, filed Mar. 14, 2013 and U.S. provisionalapplication Ser. No. 61/839,330, filed Jun. 25, 2013; all of which areincorporated by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM062159 andGM097206 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 7, 2014, isnamed 41135-720.601_SL.txt and is 20,561 bytes in size.

FIELD OF THE INVENTION

Described herein are targeting agent antibody conjugates comprising oneor more unnatural amino acids, methods of making such constructs,pharmaceutical compositions and medicaments comprising such constructs,and methods of using such constructs and compositions to prevent,inhibit, and/or treat a disease or condition in a subject.

BACKGROUND OF THE INVENTION

Antibodies are natural proteins that the vertebrate immune system formsin response to foreign substances (antigens), primarily for defenseagainst infection. For over a century, antibodies have been induced inanimals under artificial conditions and harvested for use in therapy ordiagnosis of disease conditions, or for biological research. Eachindividual antibody producing cell produces a single type of antibodywith a chemically defined composition, however, antibodies obtaineddirectly from animal serum in response to antigen inoculation actuallycomprise an ensemble of non-identical molecules (e.g., polyclonalantibodies) made from an ensemble of individual antibody producingcells.

Some antibody conjugates, such as bispecific antibodies, may bind to twoor more different antigens. A number of recombinant strategies have beendeveloped to synthesize bispecific antibodies, which include singlechain variable fragment (scFv)-derived formats such as diabodies, tandemdiabodies, BiTes (bispecific T-cell engager), and DARTs (Dual AffinityRe-Targeting), as well as immunoglobulin G (IgG)-based formats such asTriomab, DVD-Ig (Dual Variable Domain antibodies), and two-in-oneantibodies. However, bispecific antibodies can have poorpharmacokinetics and physical properties, such as immunogenicity andmanufacturing challenges. Therefore, there is a need for an improvementor alternative to such existing technology. In addition, precise controlover geometry in such a targeting moiety is desirable because thegeometry can alter binding affinity and specificity. Disclosed hereinare targeting agent antibody conjugates, and methods for producing suchconjugates, with specific geometries for optimal therapeutic efficacyand target specificity by the site-specifically coupling of an antibodyor antibody fragment to a targeting agent.

SUMMARY OF THE INVENTION

Disclosed herein are targeting agent antibody conjugates comprising: atargeting agent that binds to a target cell, wherein the targeting agentis not an antibody or antibody fragment; and an antibody or antibodyfragment that does not bind to the target cell; and one or more linkers,wherein the antibody or antibody fragment is linked to the targetingagent by the one or more linkers and wherein the antibody or antibodyfragment binds an antigen on a cytotoxic effector cell. The antibody orantibody fragment may comprise one or more unnatural amino acids. Thetargeting agent may be site-specifically linked by the one or morelinkers to the one or more unnatural amino acids of the antibody orantibody fragment.

The targeting agent antibody conjugate may be of Formula I: X-L1-Y orFormula IA: Y-L1-X, wherein: X comprises the antibody or antibodyfragment; L1 comprises the one or more linkers; and Y comprises thetargeting agent.

At least a portion of the antibody may be based on or derived from ahuman, humanized, human engineered or fully human antibody. The antibodymay be a chimeric antibody. The antibody or antibody fragment may bindan antigen on a cytotoxic effector cell and the targeting agent binds acell surface protein or a cell surface marker on a target cell. Thecytotoxic effector cell may be capable of mounting an immune response.At least a portion of the antibody or antibody fragment may bind atleast a portion of a receptor on the cytotoxic effector cell. At least aportion of the antibody or antibody fragment may bind at least a portionof a co-receptor on the cytotoxic effector cell. The receptor may be aT-cell receptor (TCR). The co-receptor may comprise a T-cellco-receptor. The co-receptor may be a CD3 T-cell co-receptor. Thecytotoxic effector cell may be a hematopoietic cell. The hematopoieticcell may be selected from a macrophage, a neutrophil, an eosinophil, anatural killer cell, a B-cell, or a T-cell. The cytotoxic effector cellmay be a cytotoxic T cell. The antibody fragment may comprise a Fabfragment. The Fab fragment may comprise an anti-CD3 Fab fragment. Theanti-CD3 Fab fragment may be UCHT1. The antibody fragment may be encodedby a sequence selected from SEQ ID NOs: 1 and 2.

The targeting agent may be selected from a small molecule, acell-targeting molecule, a ligand, a protein, a peptide, a peptoid, aDNA aptamer, a peptide nucleic acid, a vitamin a substrate or asubstrate analog. The targeting agent may not comprise an antibody or anantibody fragment. The cell surface protein or cell surface marker maybe selected from an antigen, a receptor, a co-receptor, a trans-membraneprotein or a cell marker on the target cell. The cell surface proteinmay be selected from a cholecystokinin B receptor, agonadotropin-releasing hormone receptor, a somatostatin receptor 2, anavb3 integrin, a gastrin-releasing peptide receptor, a neurokinin 1receptor, a melanocortin 1 receptor, a neurotensin receptor,neuropeptide Y receptor and C-type lectin like molecule 1. The antigenmay comprise a prostate specific membrane antigen. The targeting agentmay comprise an octreotide. The targeting agent may comprise anoctreotate. The targeting agent may comprise a somatostatin analog. Thetargeting agent may comprise a CD38 NAD+ glycohydrolase inhibitor. Thetargeting agent may comprise a pentagastrin. The targeting agent maycomprise a gonadotropin releasing hormone. The targeting agent maycomprise a CCKB antagonist. The targeting agent may comprise a cRGD. Thetargeting agent may comprise a bombesin. The targeting agent maycomprise 2-[3-(1,3-dicarboxypropy)ureidol] pentanedioic acid (DUPA). Thetargeting agent may be sufficiently small to penetrate a tumor. Thetargeting agent may be encoded by a sequence selected from SEQ ID NOs:3-40. The targeting agent antibody conjugate may further comprise asecond targeting agent. The targeting agent antibody conjugate maycomprise 1, 2, 3, 4, or more targeting agents.

The antibody or antibody fragment may bind an antigen on a cytotoxiceffector cell and the targeting agent may bind a cell surface protein ora cell surface marker on a target cell. The target cell may be acancerous cell. The cancerous cell may be derived from a prostatecancer. The cancerous cell may be derived from an epithelial cancer. Thecancerous cell may be derived from a breast cancer. The cancerous cellmay be derived from a kidney cancer. The cancerous cell may be derivedfrom a lung cancer. The cancerous cell may be derived from a coloncancer. The cancerous cell may be derived from a colorectal cancer. Thecancerous cell may be derived from a gastric cancer. The cancerous cellmay be derived from a brain cancer. The cancerous cell may be derivedfrom a glioblastoma. The cancerous cell may be derived from a pancreaticcancer. The cancerous cell may be derived from a myeloid leukemia. Thecancerous cell may be derived from a cervical cancer. The cancerous cellmay be derived from a medullary thyroid carcinoma. The cancerous cellmay be derived from a stromal ovarian cancer. The cancerous cell may bederived from an astrocytoma. The cancerous cell may be derived from anendometrial cancer. The cancerous cell may be derived from aneuroendocrine cancer. The cancerous cell may be derived from agastroenteropancreatic tumor. The cancerous cell may be derived from anon-Hodgkin's lymphoma. The cancerous cell may be derived from anexocrine pancreatic cancer. The cancerous cell may be derived from anEwing's sarcoma. The cancerous cell may be derived from a skin cancer.The skin cancer may be a melanoma. The skin cancer may be aneoangiogenic skin cancer. The lung cancer may be a small cell lungcancer.

X may be coupled to L1. Y may be coupled to L1. X may be coupled to L1by an oxime. Y may be coupled to L1 by an oxime. X may be coupled to L1by a covalent bond, ionic bond, or non-covalent bond. Y may be coupledto L1 by a covalent bond, ionic bond, or non-covalent bond. L1 mayprovide between about 10 and about 100 angstrom (A) distance between Xand Y. L1 may provide equal to or less than about 50 angstrom (A)distance between X and Y. L1 may provide equal to or less than about 45angstrom (A) distance between X and Y. L1 may provide equal to or lessthan about 40 angstrom (A) distance between X and Y. L1 may provideequal to or less than about 30 angstrom (A) distance between X and Y. L1may provide equal to or greater than about 5 angstrom (A) distancebetween X and Y. L1 may provide equal to or greater than about 10angstrom (A) distance between X and Y. L1 may provide equal to orgreater than about 15 angstrom (A) distance between X and Y. L1 mayprovide equal to or greater than about 20 angstrom (A) distance betweenX and Y. L1 may provide a distance between X and Y including and betweenabout 10 to about 100 angstrom (A).

The one or more unnatural amino acids of the antibody or antibodyfragment comprises a p-acetylphenylalanine (pAcF). The one or moreunnatural amino acids of the antibody or antibody fragment comprises aselenocysteine. The one or more unnatural amino acids may comprise (a)various substituted tyrosine and phenylalanine analogues such asO-methyl-L-tyrosine, p-amino-L-phenylalanine, 3-nitro-L-tyrosine,p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine andp-isopropyl-L-phenylalanine; (b) amino acids with aryl azide andbenzophenone groups that may be photo-cross-linked; (c) amino acids thathave unique chemical reactivity including acetyl-L-phenylalanine andm-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-(2-propynyl)-L-tyrosine,p-ethylthiocarbonyl-L-phenylalanine andp-(3-oxobutanoyl)-L-phenylalanine; (d) heavy-atom-containing amino acidsfor phasing in X-ray crystallography including p-iodo andp-bromo-L-phenylalanine; (e) the redox-active amino aciddihydroxy-L-phenylalanine; (f) glycosylated amino acids includingb-N-acetylglucosamine-O-serine and a-N-acetylgalactosamine-O-threonine;(g) fluorescent amino acids with naphthyl, dansyl, and 7-aminocoumarinside chains; (h) photocleavable and photoisomerizable amino acids withazobenzene and nitrobenzyl Cys, Ser, and Tyr side chains; (i) thephosphotyrosine mimetic p-carboxymethyl-L-phenylalanine; (j) theglutamine homologue homoglutamine; (k) 2-aminooctanoic acid; (1) or acombination of (a)-(k). The one or more unnatural amino may comprise atleast one oxime, carbonyl, dicarbonyl, hydroxylamine, cyclooctyne,aryl/alkyl azides, norbornene, cyclopropene, trans-cyclooctene, andtetrazine group. The one or more unnatural amino acids may begenetically encoded. The one or more unnatural amino acids may beincorporated into the antibody or antibody fragment. The one or moreunnatural amino acids may be site-specifically incorporated the antibodyor antibody fragment. The targeting agent antibody conjugate maycomprise two or more unnatural amino acids. The targeting agent antibodyconjugate may comprise three or more unnatural amino acids. Thetargeting agent antibody conjugate may comprise four or more unnaturalamino acids. The one or more unnatural amino acids may replace one ormore amino acid residues in the antibody or antibody fragment. The oneor more unnatural amino acids may replace an amino acid residue in aheavy chain of the antibody or antibody fragment. The one or moreunnatural amino acids of the antibody or antibody fragment replace anamino acid residue in a light chain of the antibody or antibodyfragment. The one or more unnatural amino acids of the antibody orantibody fragment replace an amino acid residue in a variable region ofthe antibody or antibody fragment.

Further disclosed herein are pharmaceutical compositions comprising atargeting agent antibody conjugate comprising: an antibody or antibodyfragment; a targeting agent, wherein the targeting agent is not anantibody or antibody fragment; and one or more linkers, wherein theantibody or antibody fragment is linked to the targeting agent by theone or more linkers. The antibody or antibody fragment and the targetingagent may be site-specifically linked. The antibody or antibody fragmentmay comprise one or more unnatural amino acids. The pharmaceuticalcomposition may further comprise a pharmaceutically acceptable diluent.The pharmaceutical composition may further comprise a pharmaceuticallyacceptable excipient. The pharmaceutical composition may furthercomprise a pharmaceutically acceptable carrier.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, comprising administering a targeting agentantibody conjugate comprising an antibody or antibody fragment; atargeting agent, wherein the targeting agent is not an antibody orantibody fragment; and one or more linkers, wherein the antibody orantibody fragment is linked to the targeting agent by the one or morelinkers. The antibody or antibody fragment and the targeting agent maybe site-specifically linked. The antibody or antibody fragment maycomprise one or more unnatural amino acids. The disease or condition maybe a pathogenic infection. The pathogenic infection may be a bacterialinfection. The pathogenic infection may be a viral infection. Thedisease or condition may be an inflammatory disease. The disease orcondition may be an autoimmune disease. The autoimmune disease may bediabetes. The disease or condition may be a cancer. The disease orcondition may be a prostate cancer. The disease or condition may be anepithelial cancer. The disease or condition may be a kidney cancer. Thedisease or condition may be lung cancer. The disease or condition may bea colon cancer. The disease or condition may be a colorectal cancer. Thedisease or condition may be a gastric cancer. The disease or conditionmay be a brain cancer. The disease or condition may be a glioblastoma.The disease or condition may be a pancreatic cancer. The disease orcondition may be a myeloid leukemia. The disease or condition may be acervical cancer. The disease or condition may be a medullary thyroidcarcinoma. The disease or condition may be a breast cancer. The diseaseor condition may be an ovarian cancer. The disease or condition may bean astrocytoma. The disease or condition may be an endometrial cancer.The disease or condition may be a neuroendocrine cancer. The disease orcondition may be a gastroenteropancreatic tumor. The disease orcondition may be a non-Hodgkin's lymphoma. The disease or condition maybe an exocrine pancreatic cancer. The disease or condition may be anEwing's sarcoma. The disease or condition may be a skin cancer. The skincancer may be a melanoma. The skin cancer may be a neoangiogenic skincancer. The lung cancer may be a small cell lung cancer. Administeringthe targeting agent antibody conjugate or pharmaceutical composition maycomprise an intravenous administration, a subcutaneous administration,an intraperitoneal administration, an intramuscular administration, anintravascular administration, an intrathecal administration, anintravitreal administration, a topical administration or an infusion.Administering may comprise oral administration. Administering maycomprise intranasal administration. Administering may comprise use of amicroneedle device.

Disclosed herein are targeting agent antibody conjugates comprising: anantibody or antibody fragment; one or more DUPA molecules; and one ormore linkers, wherein the antibody or antibody fragment is linked to theone or more DUPA molecules by the one or more linkers. The antibody orantibody fragment may be site-specifically linked to the one or moreDUPA molecules by the one or more linkers. The antibody or antibodyfragment may comprise one or more unnatural amino acids. The antibody orantibody fragment may be linked to the one or more DUPA molecules by theone or more linkers to the one or more unnatural amino acids. Theantibody or antibody fragment may alternatively be linked to the one ormore DUPA molecules by the one or more linkers to a natural amino acid.The antibody or antibody fragment may be an anti-CD3 Fab.

Disclosed herein are targeting agent antibody conjugates comprising: ananti-CD3 Fab; one or more DUPA molecules; and one or more linkers,wherein the anti-CD3 Fab is linked to the one or more DUPA molecules bythe one or more linkers. The anti-CD3 Fab may comprise one or moreunnatural amino acids. The one or more unnatural amino acids may replacea natural amino acid of the anti-CD3 Fab. The one or more unnaturalamino acids may replace a natural amino acid of the anti-CD3 Fab,wherein the natural amino acid is selected from Lysine 138 (Lys 138) ofa heavy chain of the anti-CD3 Fab, Alanine 123 (Ala 123) of a heavychain of the anti-CD3 Fab, Threonine 109 (Thr 109) of a heavy chain ofthe anti-CD3 Fab and Serine 202 (Ser 202) of a heavy chain of theanti-CD3 Fab. A first DUPA molecule and a second DUPA molecule may besite-specifically linked to a first unnatural amino acid and a secondunnatural amino acid of the anti-CD3 Fab a first linker and a secondlinker. The targeting agent antibody conjugate may be of Formula I:X-L-Y or Formula IA: Y-L-X, wherein: X comprises the anti-CD3 Fab; Lcomprises the one or more linkers; and Y comprises one or more DUPAmolecules.

The targeting agent antibody conjugate may comprise a compound selectedfrom Formula V, Formula VI, Formula VII or Formula VIII:

The targeting agent antibody conjugate may comprise a compound ofFormula IX:

The targeting agent antibody conjugate may comprise a compound ofFormula X:

The targeting agent antibody conjugate may comprise a compound ofFormula XI:

In another aspect, provided herein are compounds of Formula XII:

Y-L-A¹-L¹-A²-L²-A³-L³-X²  (Formula XII)

wherein:

-   -   Y is a ligand of prostate specific membrane antigen (PSMA);    -   L is

-   -   A¹ is selected from the group consisting of an aryl, a 5- to        6-membered heteroaryl, —C(O)—, —N(R¹)—, —O—, —C(O)N(R¹)—,        —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—, and —N(R¹)S(O)_(1,2)—;    -   L is

-   -   A² is selected from the group consisting of a bond, —C(O)—,        —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—, and        —N(R¹)S(O)_(1,2)—;    -   L² is

-   -   A³ is a bond,

-   -   L³ is

-   -   X² is a linker bound to a functional group that reacts with an        amino acid, or a linker bound to a modified amino acid, wherein        the modified amino acid is part of X, wherein X is a modified        therapeutic peptide, protein, or antibody;    -   each R¹ is independently selected from H, alkyl, or haloalkyl;    -   each R², R²¹, R²², and R²³ is independently selected from H,        halo, —OR¹, —CN, —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl,        or heteroarylalkyl;    -   each R³ is independently selected from halo, —OR¹, —CN, —SR¹,        alkyl, cycloalkyl, haloalkyl, arylalkyl, or heteroarylalkyl,        —NO₂, and NR¹R¹;    -   each G¹ and G² is independently selected from the group        consisting of a bond, —C(O)—, —N(R¹)—, —O—, —C(O)N(R¹)—,        —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—, and —N(R¹)S(O)_(1,2)—;    -   each Z, Z¹, Z², and Z³ is independently selected from the group        consisting of a bond, —O—, and —N(R¹)—;    -   k, k¹, k² and k³ are each independently selected from 0, 1, 2,        3, 4, 5, 6, 7, 8, 9, and 10;    -   m¹, m² and m³ are each independently selected from 0, 1, 2, 3,        4, 5, 6, 7, 8, 9, and 10; and    -   p is 0, 1, 2, 3 or 4;    -   or a stereoisomer thereof.

In some embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIa:

wherein:

-   -   Q is selected from the group consisting of:

and

-   -   E is selected from the group consisting of:

In some embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIb:

In further embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIc:

In some embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIId:

In further embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIe:

In still further embodiments described above or below of a compound ofFormula XII, the compound is of Formula XIIf:

In some embodiments described above or below of a compound of FormulaXII, A¹ is —C(O)N(H)—. In some embodiments described above or below of acompound of Formula XII, A¹ is

In some embodiments described above or below of a compound of FormulaXII, A³ is

In further embodiments described above or below of a compound of FormulaXII, A³ is

In some embodiments described above or below of a compound of FormulaXII, each R², R²¹, R²², R²³, and R²⁴ is independently selected from H,F, —CH₃, or —CF₃. In some embodiments described above or below of acompound of Formula XII, each R², R²¹, R²², R²³, and R²⁴ is H.

In some embodiments described above or below of a compound of FormulaXII,

-   -   X² is

-   -   wherein:        -   A⁴ is selected from the group consisting of a bond, —C(O)—,            —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—,            and —N(R¹)S(O)_(1,2)—;        -   each R²⁴ is independently selected from H, halo, —OR¹, —CN,            —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl, or            heteroarylalkyl;        -   k⁴ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;        -   Z⁴ is selected from a bond, aryl, and a 5- to 6-membered            heteroaryl; and        -   X¹ is —ONH₂,

-   -   -    —N₃,

-   -   -    —N(H)NH₂, or —SH.

In some embodiments described above or below of a compound of FormulaXII, X² is

In further embodiments described above or below of a compound of FormulaXII, X² is

In some embodiments described above or below of a compound of FormulaXII, the compound is selected from:

or a stereoisomer thereof.

In some embodiments described above or below of a compound of FormulaXII,

-   -   X² is

-   -   wherein:        -   A⁴ is selected from the group consisting of a bond, —C(O)—,            —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—,            and —N(R¹)S(O)_(1,2)—;        -   each R²⁴ is independently selected from H, halo, —OR¹, —CN,            —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl, or            heteroarylalkyl;        -   k⁴ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;        -   Z⁴ is selected from a bond, aryl, and a 5- to 6-membered            heteroaryl; and        -   X³ is

-   -   -    or —S—;        -   X is a modified therapeutic peptide, protein, or antibody;        -   L⁴ is a bond directly attached to a modified amino acid, or            a linker bound to a modified amino acid, wherein the            modified amino acid is part of X.            In some embodiments described above or below of a compound            of Formula XII, the amino acid is an unnatural amino acid.

In some embodiments described above or below of a compound of FormulaXII, k is 1, 2, or 3; and Z is a bond.

In some embodiments described above or below of a compound of FormulaXII, A¹ is —C(O)N(R¹)—, 6-membered aryl, or 5-membered heteroaryl.

In some embodiments described above or below of a compound of FormulaXII, m¹ is 0; k¹ is 6 or 7; and Z¹ is a bond.

In some embodiments described above or below of a compound of FormulaXII, A² is a bond; m² and k² are 0; and Z² is a bond.

In some embodiments described above or below of a compound of FormulaXII, A² is —C(O)N(H)—; m² is 2; k² is 2; and Z² is a bond. In someembodiments described above or below of a compound of Formula XII, A² is—C(O)N(H)—; m² is 3; k² is 2; and Z² is a bond. In some embodimentsdescribed above or below of a compound of Formula XII, A² is —C(O)N(H)—;m² is 10; k² is 2; and Z² is a bond.

In some embodiments described above or below of a compound of FormulaXII, R³ is —NO₂; and p is 2.

In some embodiments described above or below of a compound of FormulaXII, each GX¹ and GX² are independently selected from the groupconsisting of —N(H)—, —C(O)N(H)—, and —N(H)C(O)—.

In some embodiments described above or below of a compound of FormulaXII, m³ is 3; k³ is 2; and Z³ is a bond. In some embodiments describedabove or below of a compound of Formula XII,

m³ is 2; k³ is 2; and Z³ is a bond.

In some embodiments described above or below of a compound of FormulaXII, A³ is a bond; m³ and k³ are 0; and Z³ is a bond.

Further disclosed herein are compositions comprising a targeting agentantibody conjugate comprising an antibody or antibody fragment; one ormore linkers; and a targeting agent wherein the purity of the targetingagent antibody conjugate or the compound is at least 90%. The targetingagent antibody construct may comprise an unnatural amino acid. Theantibody or antibody fragment and the targeting agent may be sitespecifically linked.

Further disclosed herein are compounds comprising a Formula selectedfrom Formulas XII or XIIa-f, wherein the purity of the compound is atleast 90%.

Disclosed herein are targeting agent antibody conjugates comprising: anantibody or antibody fragment; one or more linkers; and a targetingagent, wherein the antibody or antibody fragment binds to or interactswith a receptor, co-receptor, antigen or cell marker on a first cell;and the targeting agent binds to or interacts with a receptor,co-receptor, antigen or cell marker on a second cell.

Further disclosed herein are targeting agent antibody conjugatescomprising an antibody or antibody fragment; one or more linkers; and atargeting agent, wherein the antibody or antibody fragment binds to orinteracts with a receptor, co-receptor, antigen or cell marker on afirst cell; the targeting agent binds to or interacts with a receptor,co-receptor, antigen or cell marker on a second cell; and the one ormore linkers links the antibody or antibody fragment and the targetingagent site-specifically.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, may be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown.

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings. Itis emphasized that, according to common practice, the various featuresof the drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 depicts a scheme for synthesizing a PSMA binding targetingagent-linker compound.

FIG. 2 depicts a ribbon diagram of a UCHT1-Fab fragment.

FIG. 3A depicts a scheme for linking a PSMA binding targetingagent-linker compound to a linker-conjugated Fab fragment.

FIG. 3B-3E shows examples of linkers.

FIGS. 4A and 4C depict ESI-MS analysis of Fab fragments before and afterlinker conjugation.

FIGS. 4B and 4D depict deconvoluted mass spectrum of Fab fragmentsbefore and after linker conjugation.

FIG. 5A-5B depicts flow cytometry analysis of Jurkat cells (FIG. 5A) andC4-2 cells (FIG. 5B) incubated with targeting agent antibody conjugates.

FIG. 5C depicts FACS-based binding assay of different Fabs to C4-2cells.

FIG. 6A depicts the relative fluorescence of C4-2 cells treated withdifferent concentrations of conjugated and unconjugated Fabs in C4-2cells.

FIG. 6B depicts the percent cytotoxicity of C4-2 or DU145 cellsincubated with double-Phthal or a mixture.

FIG. 6C depicts Proinflamatory cytokine TNF-alpha levels in thesupernatant from the cytotoxicity assay. Dose-dependent TNF-alphasignals were detected only in the presence of PSMA-positive C4-2 cellsby ELISA assay (R&D systems).

FIG. 7A depicts a schematic of an in vivo xenograft model experiment

FIG. 7B depicts the tumor volume of C4-2 cells implanted in mice treatedwith PBS, conjugated Fab and PBMC, or conjugated Fab (no PBMC).

FIG. 8 depicts the structure of p-acetylphenylaline.

FIG. 9 depicts High-throughput FACS analysis of the binding of differentP-anti-CD3 conjugates to PSMA-positive C4-2 cells at variousconcentrations. P-Phthal-double conjugate shows significantly improvedaffinity compared to the corresponding monoconjugates.

FIG. 10 depicts In vitro cytotoxicity of target cells in the presence offreshly purified unactivated hPBMCs (T:E=1:10). Conjugates showdose-dependent activity on PSMA-positive C4-2 cells with different EC₅₀values whereas the unconjugated mixture shows no activity.

FIG. 11 depicts Microscope images of the 100 pM wells, where cellclusters may be observed only on C4-2 cells in the presence of theP-targeting agent antibody conjugate conjugate. Unconjugated anti-CD3and P-Phthal mixture had no effect on cell clustering.

FIG. 12 depicts in vivo efficacy studies of P-targeting agent antibodyconjugate. In the prophylactic model, 1×10⁶ C4-2 cells were mixed with2×10⁶ PBMCs (1:2 ratio) in Matrigel and injected SC into the rightshoulder of male NOD-SCID mice. P-targeting agent antibody conjugate,unconjugated Fab, or PBS (n=6) were administered at 2 mg/kg everyday for10 days by IV starting on Day 4. Tumors were monitored by externalcaliper measurements for regular intervals for 6 weeks. P-targetingagent antibody conjugate suppressed tumor growth (p<0.0001) while thecontrol groups developed rapid tumors. * indicates that mice with largetumors (>1000 mm³) were sacrificed before these days.

FIG. 13 depicts the design of the second-generation PSMA targetingcompound, P-TriA. The amide group at C-9 position in P-Phthal waschanged to a triazole group to increase the affinity with a hydrophobicpocket near the PSMA enzyme active site. Based on the distance betweenthe first and second hydrophobic binding pocket (˜11 Å) in the crystalstructure, shorter hydrocarbon linker was introduced before thephthalimide group.

FIG. 14 depicts the chemical structures of P-Tet, P-Und, and P-DNP.

FIG. 15A-J depict reaction schemes for the synthesis of(S)-2-(3-((S)-5-(4-(6-(4-(1-(aminooxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-ylcarbamoyl)benzamido)hexyl)-1H-1,2,3-triazol-1-yl)-1-carboxypentyl)ureido)pentanedioicacid (P-TriA).

FIG. 15K depicts the ESI-MS analysis P-TriA conjugated to an anti-CD3Fab.

FIG. 15L depicts the deconvoluted mass spectrum of P-TriA conjugated toan anti-CD3 Fab

FIG. 16A-B depict the results of a PSMA inhibition assay. (A) depictsrepresentative PSMA inhibition curves by different inhibitors. (B)depicts a Km curve obtained from PSMA inhibition assay.

FIG. 17A-B depict the results of in vitro cytotoxicity assays in C4-2cells at 24 hours and 48 hours.

FIG. 18A-F depict structures of exemplary targeting agents.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. Examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein may be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method may be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Disclosed herein are targeting agent antibody conjugates comprising: atargeting agent that binds to a target cell, wherein the targeting agentis not an antibody or antibody fragment; and an antibody or antibodyfragment that does not bind to the target cell; and one or more linkers,wherein the antibody or antibody fragment is linked to the targetingagent by the one or more linkers and wherein the antibody or antibodyfragment binds an antigen on a cytotoxic effector cell. The antibody orantibody fragment may comprise one or more unnatural amino acids. Thetargeting agent may be site-specifically linked by the one or morelinkers to the one or more unnatural amino acids of the antibody orantibody fragment.

Further disclosed herein are targeting agent antibody conjugates. Thetargeting agent antibody conjugate may comprise Formula I: X-L1-Y orFormula IA: Y-L1-X, wherein (a) X comprises at least a portion ofantibody or antibody fragment; (b) L1 comprises one or more linkers; and(c) Y comprises a targeting agent, wherein the antibody or antibodyfragment is linked to the targeting agent by the one or more linkers.The antibody or antibody fragment and the targeting agent may besite-specifically linked. Generally X binds a cytotoxic effector celland Y binds a target cell. For example, X may bind an antigen on acytotoxic T cell and Y may bind a receptor or cell surface marker on acancer cell. Another example is a cytotoxic effector cell may be amacrophage and the target cell may be a pathogen.

Disclosed herein are targeting agent antibody conjugates. The targetingagent antibody conjugate may comprise Formula I: X-L1-Y or Formula IA:Y-L1-X, wherein (a) X comprises at least a portion of antibody orantibody fragment; (b) L¹ comprises one or more linkers; and (c) Ycomprises a targeting agent, wherein the antibody or antibody fragmentis linked to the targeting agent by the one or more linkers and whereinthe antibody or antibody fragment and the targeting agent aresite-specifically linked. Generally, the targeting agent antibodyconjugate comprises one or more unnatural amino acids. Generally, theantibody or antibody fragment comprises the one or more unnatural aminoacids.

Further disclosed herein are targeting antibody conjugates of theFormula I: X-L1-Y or Formula IA: Y-L1-X, wherein X comprises more thanone antibody or antibody fragment. X may comprise 1, 2, 3, 4 or moreantibodies or antibody fragments. X may comprise two antibodies orantibody fragments. Two or more antibodies or antibody fragments may belinked by a peptide. The peptide may be about 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acids in length. The peptide may be about five amino acids inlength. X may comprise one or more single chain variable fragments(scFvs). X may comprise one or more Fabs. X may comprise 1, 2, 3, 4 ormore scFvs. X may comprise 1, 2, 3, 4 or more Fabs. X may comprise amonovalent Fab. X may comprise a bivalent Fab. X may comprise atrivalent Fab. X may comprise a tetravalent Fab. X may comprise amonovalent scFv. X may comprise a bivalent scFv. X may comprise atrivalent scFv. X may comprise a tetravalent scFv. One or more Fabs maybe the same. One or more Fabs may be different. One or more scFvs may bethe same. One or more scFvs may be different.

Further disclosed herein are targeting antibody conjugates of theFormula I: X-L1-Y or Formula IA: Y-L1-X, wherein X comprises more thanone antibody or antibody fragment and Y comprises one or more targetingagents. Y may comprise 1, 2, 3, 4 or more targeting agents. The one ormore targeting agents may be the same. The one or more targeting agentsmay be different. The targeting antibody conjugate may comprise a secondlinker wherein the first linker links one targeting agent to the firstantibody or antibody fragment and the second linker links the secondtargeting agent to the second antibody or antibody fragment. Thetargeting agent may comprise 2, 3, 4 or more linkers wherein each linkerlinks each targeting agent to an antibody or antibody fragment of thetargeting antibody conjugate.

Further disclosed herein are targeting antibody conjugates of theFormula I: X-L¹-Y or Formula IA: Y-L1-X, wherein X comprises oneantibody or antibody fragment and wherein Y comprises one or moretargeting agents. Y may comprise 1, 2, 3, 4, 5, 6 or more targetingagents. The one or more targeting agents may be the same. The one ormore targeting agents may be different. The targeting antibody conjugatemay comprise 1, 2, 3, 4, 5, 6 or more linkers wherein each linker linksa targeting agent to the antibody or antibody fragment.

The efficacy of the targeting antibody conjugates may be optimized bymodifying linker structure, linker length, relative binding orientationand stoichiometry of the targeting agent. Conventional chemicalapproaches that use lysine or cysteine chemistry to link antigen-bindingmoieties tend to yield heterogeneous products which likely differ intheir ability to accommodate productive geometries for the formation ofimmunological synapses and/or have reduced stability or half-life invivo. Circumventing these challenges, are targeting antigen antibodyconjugates disclosed herein that incorporate unnatural amino acids whichallow for precise control of linker moiety placement and thus precisecontrol over targeting agent antibody conjugate geometry as well asgeneration of homogenous pools of targeting agent antibody conjugates.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds a prostate specific membrane antigen (PSMA).The targeting agent may be DUPA. The antibody fragment may be ananti-CD3 Fab. The anti-CD3 Fab may be UCHT1. The linker may be a P-TriAlinker. The linker may be a P-Und linker. The linker may be a P-Tetlinker. The linker may be a P-Phthal linker. The linker may be a P-DNPlinker. The targeting agent antibody conjugate may further comprise asecond anti-CD3 Fab. The targeting agent antibody conjugate may furthercomprise a second, third, or fourth targeting agent that binds aprostate specific membrane antigen. The second, third, or fourthtargeting agent that binds a prostate specific membrane antigen maycomprise DUPA. The targeting agent antibody conjugate may comprise abivalent anti-CD3 Fab and four DUPAs, wherein a first and a second DUPAare linked by a first linker and a second linker to a first Fab of theanti-CD3 Fab and a third and a fourth DUPA are linked by a third linkerand a fourth linker to a second Fab of the anti-CD3 Fab. The anti-CD3antibody or antibody fragment may comprise one or more unnatural aminoacids. The anti-CD3 antibody or antibody fragment may comprise one ormore unnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to DUPA. DUPA may besite-specifically linked to the anti-CD3 antibody or antibody fragmentat the unnatural amino acid of the anti-CD3 antibody or antibodyfragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds a c-type lectin-like molecule. The anti-CD3antibody or antibody fragment may comprise one or more unnatural aminoacids. The anti-CD3 antibody or antibody fragment may comprise one ormore unnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to the targeting agent thatbinds the c-type lectin-like molecule. The targeting agent that bindsc-type lectin-like molecule may be site-specifically linked to theanti-CD3 antibody or antibody fragment at the unnatural amino acid ofthe anti-CD3 antibody or antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds a cholecystokinin B receptor (CCKBR). Thetargeting agent that binds the cholecystokinin B receptor may comprisepentagastrin. The targeting agent that binds the cholecystokinin Breceptor may comprise a CCKBR antagonist. The anti-CD3 antibody orantibody fragment may comprise one or more unnatural amino acids. Theanti-CD3 antibody or antibody fragment may comprise one or moreunnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to pentagastrin. The anti-CD3antibody or antibody fragment may be site-specifically linked to theCCKBR antagonist. The CCKBR antagonist may be site-specifically linkedto the anti-CD3 antibody or antibody fragment at the unnatural aminoacid of the anti-CD3 antibody or antibody fragment. Pentagastrin may besite-specifically linked to the anti-CD3 antibody or antibody fragmentat the unnatural amino acid of the anti-CD3 antibody or antibodyfragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds a gonadotropin releasing hormone receptor(GnRHR). The targeting agent that binds the GnRHR receptor may comprisegonadotropin releasing hormone (GnRH). The anti-CD3 antibody or antibodyfragment may comprise one or more unnatural amino acids. The anti-CD3antibody or antibody fragment may comprise one or more unnatural aminoacids, wherein the unnatural amino acid is site-specificallyincorporated. The anti-CD3 antibody or antibody fragment may besite-specifically linked to GnRH. GnRH may be site-specifically linkedto the anti-CD3 antibody or antibody fragment at the unnatural aminoacid of the anti-CD3 antibody or antibody fragment

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds a somatostatin receptor 2 (SST2). Thetargeting agent that binds the somatostatin receptor 2 may compriseoctreotide. The targeting agent that binds the somatostatin receptor 2may comprise octreotate. The targeting agent that binds the somatostatinreceptor 2 may comprise a somatostatin anolog. The anti-CD3 antibody orantibody fragment may comprise one or more unnatural amino acids. Theanti-CD3 antibody or antibody fragment may comprise one or moreunnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to octreotide, octreotate orthe somatostatin analog. The octreotide, octreotate or somatostatinanalog may be site-specifically linked to the anti-CD3 antibody orantibody fragment at the unnatural amino acid of the anti-CD3 antibodyor antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds an avb3 integrin. The targeting agent thatbinds the avb3 integrin may comprise a cyclic Arginine-Glycine-AsparticAcid peptide (cRGD). The anti-CD3 antibody or antibody fragment maycomprise one or more unnatural amino acids. The anti-CD3 antibody orantibody fragment may comprise one or more unnatural amino acids,wherein the unnatural amino acid is site-specifically incorporated. Theanti-CD3 antibody or antibody fragment may be site-specifically linkedto cRGD. The cRGD may be site-specifically linked to the anti-CD3antibody or antibody fragment at the unnatural amino acid of theanti-CD3 antibody or antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds a gastrin releasing peptide receptor. Thetargeting agent that binds the gastrin releasing peptide receptor maycomprise a bombesin. The anti-CD3 antibody or antibody fragment maycomprise one or more unnatural amino acids. The anti-CD3 antibody orantibody fragment may comprise one or more unnatural amino acids,wherein the unnatural amino acid is site-specifically incorporated. Theanti-CD3 antibody or antibody fragment may be site-specifically linkedto bombesin. The bombesin may be site-specifically linked to theanti-CD3 antibody or antibody fragment at the unnatural amino acid ofthe anti-CD3 antibody or antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds neurokinin 1 receptor. The anti-CD3 antibodyor antibody fragment may comprise one or more unnatural amino acids. Theanti-CD3 antibody or antibody fragment may comprise one or moreunnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to the targeting agent thatbinds the neurokinin 1 receptor. The targeting agent that bindsneurokinin 1 receptor may be site-specifically linked to the anti-CD3antibody or antibody fragment at the unnatural amino acid of theanti-CD3 antibody or antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds melanocortin 1 receptor. The anti-CD3antibody or antibody fragment may comprise one or more unnatural aminoacids. The anti-CD3 antibody or antibody fragment may comprise one ormore unnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to the targeting agent thatbinds the melanocortin 1 receptor. The targeting agent that bindsmelanocortin 1 receptor may be site-specifically linked to the anti-CD3antibody or antibody fragment at the unnatural amino acid of theanti-CD3 antibody or antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds neurotensin receptor. The anti-CD3 antibodyor antibody fragment may comprise one or more unnatural amino acids. Theanti-CD3 antibody or antibody fragment may comprise one or moreunnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to the targeting agent thatbinds the neurotensin receptor. The targeting agent that bindsneurotensin receptor may be site-specifically linked to the anti-CD3antibody or antibody fragment at the unnatural amino acid of theanti-CD3 antibody or antibody fragment.

Disclosed herein is a targeting agent antibody conjugate comprising ananti-CD3 antibody or antibody fragment; one or more linkers; and atargeting agent that binds neuropeptide Y receptor. The anti-CD3antibody or antibody fragment may comprise one or more unnatural aminoacids. The anti-CD3 antibody or antibody fragment may comprise one ormore unnatural amino acids, wherein the unnatural amino acid issite-specifically incorporated. The anti-CD3 antibody or antibodyfragment may be site-specifically linked to the targeting agent thatbinds the neuropeptide Y receptor. The targeting agent that bindsneuropeptide Y rceptor may be site-specifically linked to the anti-CD3antibody or antibody fragment at the unnatural amino acid of theanti-CD3 antibody or antibody fragment.

Relative to bispecific antibodies with similar binding targets, thetargeting agent antibody conjugates disclosed herein, may show animproved serum half-life, selectivity and potency.

Further disclosed herein are methods of producing targeting agentantibody conjugates, These methods allow for facile generation ofvarious targeting agent antibody conjugates with different relativegeometries.

I. Antibodies, Antibody Fragments, and Targeting Agents

The targeting agent antibody conjugate may comprise an Ig-targetingagent construct, wherein X comprises an immunoglobulin and Y comprises atargeting agent. The targeting agent antibody conjugate may comprise aFab-targeting agent construct, wherein X comprises a Fab fragment and Ycomprises a targeting agent. X and Y may be linked by one or morelinkers (e.g., L¹, L²). As used herein, the term “antibody fragment”refers to any form of an antibody other than the full-length form.Antibody fragments herein include antibodies that are smaller componentsthat exist within full-length antibodies, and antibodies that have beenengineered. Antibody fragments include, but are not limited to, Fv, Fc,Fab, and (Fab′)2, single chain Fv (scFv), diabodies, triabodies,tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3,combinations of CDRs, variable regions, framework regions, constantregions, heavy chains, light chains, alternative scaffold non-antibodymolecules, and bispecific antibodies. Unless specifically notedotherwise, statements and claims that use the term “antibody” or“antibodies” may specifically include “antibody fragment” and “antibodyfragments.”

The antibodies disclosed herein may be human, fully human, humanized,human engineered, non-human, and/or chimeric antibody. For example, theantibody of Formula I may be a humanized antibody. In another example,the antibody of Formula I is a chimeric antibody. The antibodiesdisclosed herein may be based on or derived from human, fully human,humanized, human engineered, non-human and/or chimeric antibodies. Forexample, X of Formula IA may be based on or derived from a humanengineered antibody. Alternatively, X of Formula IA may be based on orderived from a non-human antibody. The non-human antibody may behumanized to reduce immunogenicity to humans, while retaining thespecificity and affinity of the parental non-human antibody. Generally,a humanized antibody comprises one or more variable domains in whichCDRs (or portions thereof) are derived from a non-human antibody, andFRs (or portions thereof) are derived from human antibody sequences. Ahumanized antibody optionally also comprises at least a portion of ahuman constant region. In some embodiments, some FR residues in ahumanized antibody are substituted with corresponding residues from anon-human antibody (e.g., the antibody from which the CDR residues arederived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005); Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling); and Studnicka et al., U.S. Pat. No. 5,766,886.

Chimeric antibodies may refer to antibodies created through the joiningof two or more antibody genes which originally encoded for separateantibodies. A chimeric antibody may comprise at least one amino acidfrom a first antibody and at least one amino acid from a secondantibody, wherein the first and second antibodies are different. X(e.g., antibody of Formula I, IA, II) may be a chimeric antibody. Atleast a portion of the antibody or antibody fragment may be from abovine species, a human species, or a murine species. At least a portionof the antibody or antibody fragment may be from a cow. At least aportion of the antibody or antibody fragment may be from a rat, a goat,a guinea pig or a rabbit. At least a portion of the antibody or antibodyfragment may be from a human. At least a portion of the antibody orantibody fragment antibody may be from cynomolgus monkey.

The antibodies disclosed herein may be based on or derived from anantibody or antibody fragment from a mammal, bird, fish, amphibian,reptile. Mammals include, but are not limited to, carnivores, rodents,elephants, marsupials, rabbits, bats, primates, seals, anteaters,cetaceans, odd-toed ungulates and even-toed ungulates. The mammal may bea human, non-human primate, mouse, sheep, cat, dog, cow, horse, goat, orpig.

Birds include, but are not limited to, albatrosses, hummingbirds,eagles, ostriches, cardinals, kiwis, and penguins. Fish may becartilaginous fishes, ray-finned fishes, or lobe-fined fishes.Amphibians may include, but are not limited to, newts, salamanders,frogs and toads. Examples of reptiles include, but are not limited to,turtles, squamates, crocodiles and tuataras. Squamates may includeamphisbaenas, lizards and snakes.

The antibodies disclosed herein may be cross-species reactive. Forexample, an antibody may recognize a human antigen and a cynomogolusmonkey antigen (e.g. human/cyno antibody.

The antibody or antibody fragment may comprise at least a portion of asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 50% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 60% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 70% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 80% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 50% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 85% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 90% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 95% identical to asequence selected from SEQ ID NOs: 1-2. The antibody or antibodyfragment may comprise a sequence that is at least 97% identical to asequence selected from SEQ ID NOs: 1-2.

The antibody or antibody fragment may comprise a sequence comprisingfive or more amino acids based on or derived from a sequence selectedfrom SEQ ID NOs: 1-2. The antibody or antibody fragment may comprise asequence comprising 6, 7, 8, 9, 10 or more amino acids based on orderived from a sequence selected from SEQ ID NOs: 1-2. The antibody orantibody fragment may comprise a sequence comprising 15, 16, 17, 18, 19,20 or more amino acids based on or derived from a sequence selected fromSEQ ID NOs: 1-2. The antibody or antibody fragment may comprise asequence comprising 25, 30, 35, 40, 45, 50 or more amino acids based onor derived from a sequence selected from SEQ ID NOs: 1-2. The antibodyor antibody fragment may comprise a sequence comprising 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more amino acids based on or derived from asequence selected from SEQ ID NOs: 1-2. The amino acids may beconsecutive. The amino acids may be non-consecutive.

IA. Antibody or Antibody Fragments of X

The antibodies disclosed herein may comprise X, wherein X comprises atleast a portion of an antibody or antibody fragment. The antibody orantibody fragment may comprise one or more unnatural amino acids. X maycomprise an entire antibody. X may comprise at least a portion ofantibody. X may comprise at least a portion of a monoclonal antibody. Xmay comprise at least a portion of a polyclonal antibody. X may compriseat least a portion of a multivalent antibody.

X may comprise at least a portion of an antibody. The portion of theantibody may comprise an antibody fragment. The portion of the antibodymay be an immunogloblulin (Ig). The immunoglobulin may selected from anIgG, an IgA, an IgD, an IgE, an IgM, a fragment thereof or amodification thereof. The immunoglobulin may be IgG. The IgG may beIgG1. The IgG may be IgG2. The IgG may have Fc mutations for reduced FcRbinding. The Fc mutations in the IgG1 may be L234A and L235A. The Fcmutations in the IgG1 may be L234A and L235E. The Fc mutation in theIgG1 mayb be N297A. The Fc mutation in the IgG2 may be V234A and V237A.Antibody fragments include, but are not limited to, Fv, Fc, Fab, and(Fab′)2, single chain Fv (scFv), diabodies, triabodies, tetrabodies,bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR's,variable regions, framework regions, constant regions, heavy chains,light chains, alternative scaffold non-antibody molecules, andbispecific antibodies. X may comprise a Fab fragment. X may comprise atleast a portion of a heavy chain (HC) of an antibody. X may comprise atleast a portion of a light chain (LC) of an antibody. X may comprise atleast a portion of a variable region of an antibody. X may comprise atleast a portion of a constant region of an antibody.

X may comprise at least a portion of a sequence selected from SEQ IDNOs: 1-2. X may comprise a sequence that is at least 50% identical to asequence selected from SEQ ID NOs: 1-2. X may comprise a sequence thatis at least 60% identical to a sequence selected from SEQ ID NOs: 1-2. Xmay comprise a sequence that is at least 70% identical to a sequenceselected from SEQ ID NOs: 1-2. X may comprise a sequence that is atleast 80% identical to a sequence selected from SEQ ID NOs: 1-2. X maycomprise a sequence that is at least 50% identical to a sequenceselected from SEQ ID NOs: 1-2. X may comprise a sequence that is atleast 85% identical to a sequence selected from SEQ ID NOs: 1-2. X maycomprise a sequence that is at least 90% identical to a sequenceselected from SEQ ID NOs: 1-2. X may comprise a sequence that is atleast 95% identical to a sequence selected from SEQ ID NOs: 1-2. X maycomprise a sequence that is at least 97% identical to a sequenceselected from SEQ ID NOs: 1-2.

X may comprise a sequence comprising five or more amino acids based onor derived from a sequence selected from SEQ ID NOs: 1-2. X may comprisea sequence comprising 6, 7, 8, 9, 10 or more amino acids based on orderived from a sequence selected from SEQ ID NOs: 1-2. X may comprise asequence comprising 15, 16, 17, 18, 19, 20 or more amino acids based onor derived from a sequence selected from SEQ ID NOs: 1-2. X may comprisea sequence comprising 25, 30, 35, 40, 45, 50 or more amino acids basedon or derived from a sequence selected from SEQ ID NOs: 1-2. X maycomprise a sequence comprising 55, 60, 65, 70, 75, 80, 85, 90, 95, 100or more amino acids based on or derived from a sequence selected fromSEQ ID NOs: 1-2. The amino acids may be consecutive. The amino acids maybe non-consecutive.

X may comprise an antibody or antibody fragment that binds to at least aportion of a receptor on a cell. X may comprise an antibody or antibodyfragment that binds to at least a portion of a co-receptor on a cell. Xmay comprise an antibody or antibody fragment that binds to at least aportion of an antigen or cell surface marker on a cell. The cell may bea hematopoietic cell. The hematopoietic cell may be a myeloid cell. Themyeloid cell may be an erythrocyte, thrombocyte, monocyte, eosinophil,basophil, or mast cell. The hematopoietic cell may be a lymphoid cell.The hematopoietic cell may be a macrophage. The hematopoietic cell maybe a neutrophil. The lymphoid cell may be a B-cell, T-cell, or NK-cell.The hematopoietic cell may be a leukocyte. The hematopoietic cell may bea lymphocyte. The T-cell may be a cytotoxic T-cell, a helper T-cell, aregulatory T-cell, a memory T cell or a natural killer T-cell. TheT-cell may be a cytotoxic T-cell. The T-cell may be a killer T-cell.

X may comprise at least a portion of an anti-T cell receptor antibody. Xmay comprise at least a portion of an anti-T cell co-receptor antibody.X may comprise at least a portion of an antibody that binds to anantigen on a T cell. X may comprise at least a portion of an antibodythat binds to a cell surface protein on a T cell. X may comprise atleast a portion of an antibody that binds to a cell surface marker on aT cell. X may comprise at least a portion of an antibody that binds to acluster of differentiation protein on a T cell. X may comprise at leasta portion of an anti-CD3 antibody. X may comprise an anti-CD3 antibody.The anti-CD3 antibody may be UCHT1. X may comprise at least a portion ofa Fab fragment of an anti-CD3 antibody. X may comprise an antibodyfragment of an anti-CD3 antibody. X may comprise a human/cynonolguscross-reactive antiCD3 Fab (e.g. SP34).

X may comprise an antibody or antibody fragment that binds to at least aportion of a receptor on a cell. X may comprise an antibody or antibodyfragment that binds to at least a portion of a co-receptor on a cell. Xmay comprise an antibody or antibody fragment that binds to at least aportion of an antigen or cell surface marker on a cell. The cell may bean immune cell. The cell may be a hematopoietic cell. The hematopoieticcell may be a myeloid cell. The myeloid cell may be an erythrocyte,thrombocyte, neutrophil, monocyte, macrophage, eosinophil, basophil, ormast cell. The hematopoietic cell may be a lymphoid cell. The lymphoidcell may be a B-cell, T-cell, or NK-cell. The hematopoietic cell may bea leukocyte. The hematopoietic cell may be a lymphocyte. The cell may bea genetically modified cell. The cell may be genetically modified tohave cytotoxic activity. The cell may be genetically modified to haveenhanced cytotoxic activity. The cell may be modified to have decreasedcytotoxic activity.

X may comprise an antibody or antibody fragment that binds to at least aportion of a receptor on a T-cell. The receptor may be a T-cell receptor(TCR). The TCR may comprise TCR alpha, TCR beta, TCR gamma and/or TCRdelta. The receptor may be a T-cell receptor zeta.

X may comprise an antibody or antibody fragment that binds to at least aportion of a receptor on a lymphocyte, B-cell, macrophage, monocytes,neutrophils and/or NK cells. The receptor may be an Fc receptor. The Fcreceptor may be an Fc-gamma receptor, Fc-alpha receptor and/orFc-epsilon receptor. Fc-gamma receptors include, but are not limited to,FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a) andFcγRIIIB (CD16b). Fc-alpha receptors include, but are not limited to,FcaRI. Fc-epsilon receptors include, but are not limited to, FccRI andFccRII. The receptor may be CD89 (Fc fragment of IgA receptor or FCAR).The targeting agent antibody conjugate may bind specifically topathogenic bacteria or fungi when the targeting agent antibody conjugatecomprises a Fc receptor-binding antibody.

X may comprise an antibody or antibody fragment that binds at least aportion of a co-receptor on a T-cell. The co-receptor may be a CD3, CD4,and/or CD8. X may comprise an antibody fragment that binds to a CD3co-receptor. The CD3 co-receptor may comprise CD3-gamma, CD3-deltaand/or CD3-epsilon. CD8 may comprise CD8-alpha and/or CD8-beta chains.

X may comprise an antibody or at least a portion of an antibody that isa human, fully human, humanized, human engineered, non-human, orchimeric antibody. X may comprise an antibody or at least a portion ofan antibody that is a mammalian antibody. X may comprise an antibody orat least a portion of an antibody that is a non-mammalian antibody.

X may comprise a sequence based on or derived from one or moreantibodies and/or antibody fragment sequences. X may comprise a sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more homologous to a sequence based on or derivedfrom one or more antibodies and/or antibody fragments. X may comprise asequence that is at least about 70% homologous to a sequence based on orderived from one or more antibodies and/or antibody fragments. X maycomprise a sequence that is at least about 80% homologous to a sequencebased on or derived from one or more antibodies and/or antibodyfragments. X may comprise a sequence that is at least about 90%homologous to a sequence based on or derived from one or more antibodiesand/or antibody fragments. X may comprise a sequence that is at leastabout 95% homologous to a sequence based on or derived from one or moreantibodies and/or antibody fragments. The sequence may be a peptidesequence. Alternatively, the sequence is a nucleotide sequence.

X may comprise a peptide sequence that differs from a peptide sequencebased on or derived from one or more antibodies and/or antibodyfragments by less than or equal to about 20, 17, 15, 12, 10, 8, 6, 5, 4or fewer amino acids. X may comprise a peptide sequence that differsfrom a peptide sequence based on or derived from one or more antibodiesand/or antibody fragments by less than or equal to about 4 or feweramino acids. X may comprise a peptide sequence that differs from apeptide sequence based on or derived from one or more antibodies and/orantibody fragments by less than or equal to about 3 or fewer aminoacids. X may comprise a peptide sequence that differs from a peptidesequence based on or derived from one or more antibodies and/or antibodyfragments by less than or equal to about 2 or fewer amino acids. X maycomprise a peptide sequence that differs from a peptide sequence basedon or derived from one or more antibodies and/or antibody fragments byless than or equal to about 1 or fewer amino acids. The amino acids maybe consecutive, nonconsecutive, or a combination thereof. For example, Xmay comprise a peptide sequence that differs from a peptide sequencebased on or derived from one or more antibodies and/or antibodyfragments by less than about 3 consecutive amino acids. Alternatively,or additionally, X may comprise a peptide sequence that differs from apeptide sequence based on or derived from one or more antibodies and/orantibody fragments by less than about 2 non-consecutive amino acids. Inanother example, X may comprise a peptide sequence that differs from apeptide sequence based on or derived from one or more antibodies and/orantibody fragments by less than about 5 amino acids, wherein 2 of theamino acids are consecutive and 2 of the amino acids arenon-consecutive.

X may comprise a nucleotide sequence that differs from a nucleotidesequence based on or derived from one or more antibodies and/or antibodyfragments by less than or equal to about 30, 25, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or fewer nucleotides or base pairs.X may comprise a nucleotide sequence that differs from a nucleotidesequence based on or derived from one or more antibodies and/or antibodyfragments by less than or equal to about 15 or fewer nucleotides or basepairs. X may comprise a nucleotide sequence that differs from anucleotide sequence based on or derived from one or more antibodiesand/or antibody fragments by less than or equal to about 12 or fewernucleotides or base pairs. X may comprise a nucleotide sequence thatdiffers from a nucleotide sequence based on or derived from one or moreantibodies and/or antibody fragments by less than or equal to about 9 orfewer nucleotides or base pairs. X may comprise a nucleotide sequencethat differs from a nucleotide sequence based on or derived from one ormore antibodies and/or antibody fragments by less than or equal to about6 or fewer nucleotides or base pairs. X may comprise a nucleotidesequence that differs from a nucleotide sequence based on or derivedfrom one or more antibodies and/or antibody fragments by less than orequal to about 4 or fewer nucleotides or base pairs. X may comprise anucleotide sequence that differs from a nucleotide sequence based on orderived from one or more antibodies and/or antibody fragments by lessthan or equal to about 3 or fewer nucleotides or base pairs. X maycomprise a nucleotide sequence that differs from a nucleotide sequencebased on or derived from one or more antibodies and/or antibodyfragments by less than or equal to about 2 or fewer nucleotides or basepairs. X may comprise a nucleotide sequence that differs from anucleotide sequence based on or derived from one or more antibodiesand/or antibody fragments by less than or equal to about 1 or fewernucleotides or base pairs. The nucleotides or base pairs may beconsecutive, nonconsecutive, or a combination thereof. For example, Xmay comprise a nucleotide sequence that differs from a nucleotidesequence based on or derived from one or more antibodies and/or antibodyfragments by less than about 3 consecutive nucleotides or base pairs.Alternatively, or additionally, X may comprise a nucleotide sequencethat differs from a nucleotide sequence based on or derived from one ormore antibodies and/or antibody fragments by less than about 2non-consecutive nucleotides or base pairs. In another example, X maycomprise a nucleotide sequence that differs from a nucleotide sequencebased on or derived from one or more antibodies and/or antibodyfragments by less than about 5 nucleotides or base pairs, wherein 2 ofthe nucleotides or base pairs are consecutive and 2 of the nucleotidesor base pairs are non-consecutive.

The peptide sequence of X may differ from the peptide sequence of theantibody or antibody fragment that it is based on and/or derived from byone or more amino acid substitutions. The peptide sequence of X maydiffer from the peptide sequence of the antibody or antibody fragmentthat it is based on and/or derived from by two or more amino acidsubstitutions. The peptide sequence of X may differ from the peptidesequence of the antibody or antibody fragment that it is based on and/orderived from by three or more amino acid substitutions. The peptidesequence of X may differ from the peptide sequence of the antibody orantibody fragment that it is based on and/or derived from by four ormore amino acid substitutions. The peptide sequence of X may differ fromthe peptide sequence of the antibody or antibody fragment that it isbased on and/or derived from by five or more amino acid substitutions.The peptide sequence of X may differ from the peptide sequence of theantibody or antibody fragment that it is based on and/or derived from bysix or more amino acid substitutions. The peptide sequence of X maydiffer from the peptide sequence of the antibody or antibody fragmentthat it is based on and/or derived from by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 15, 17, 20, 25 or more amino acid substitutions.

The nucleotide sequence of X may differ from the nucleotide sequence ofthe antibody or antibody fragment that it is based on and/or derivedfrom by one or more nucleotide and/or base pair substitutions. Thenucleotide sequence of X may differ from the nucleotide sequence of theantibody or antibody fragment that it is based on and/or derived from bytwo or more nucleotide and/or base pair substitutions. The nucleotidesequence of X may differ from the nucleotide sequence of the antibody orantibody fragment that it is based on and/or derived from by three ormore nucleotide and/or base pair substitutions. The nucleotide sequenceof X may differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by four or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by five or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by six or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by nine or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by twelve or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by fifteen or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by eighteen or morenucleotide and/or base pair substitutions. The nucleotide sequence of Xmay differ from the nucleotide sequence of the antibody or antibodyfragment that it is based on and/or derived from by 20, 22, 24, 25, 27,30 or more nucleotide and/or base pair substitutions.

X may comprise one or more unnatural amino acids. X may comprise two ormore unnatural amino acids. X may comprise three or more unnatural aminoacids. X may comprise four or more unnatural amino acids. X may comprise5, 6, 7, 8, 9, 10 or more unnatural amino acids. X may comprise one ormore antibodies or antibody fragments, wherein one or more of theantibodies or antibody fragments comprises one or more unnatural aminoacids. X may comprise one or more unnatural amino acids, wherein oneamino acid replaces Lysine 138 of a heavy chain of the antibody orantibody fragment. X may comprise one or more unnatural amino acidsdistal to the antigen binding site of the antibody or antibody fragment.X may comprise one or more unnatural amino acids near the antigenbinding site of the antibody or antibody fragment. X may comprise one ormore unnatural amino acids in the antigen binding site of the antibodyor antibody fragment.

X may be coupled to one or more linkers. X may be linked to Y by one ormore linkers. X may be linked to Y by two or more linkers. X may belinked to Y by three or more linkers.

IB. Targeting Agents of Y

Y may comprise a targeting agent. Y may not be an antibody or anantibody fragment. Y may be selected from a small molecule, acell-targeting molecule, a ligand, a protein, a peptide, a peptoid, aDNA aptamer, a peptide nucleic acid (PNA), a vitamin, a substrate or asubstrate analog. The peptide may comprise a cyclic a peptide or alinear peptide. Y may comprise a ligand. Y may comprise at least aportion of a ligand. The ligand may be a chemical ligand. The ligand maybe a hormonal ligand. The ligand may be a peptide ligand. The ligand maybe a protein ligand. Y may be chemically modified. Y may be derivatized(e.g. with a naturally occurring protein or peptide). Y may be a smallmolecule. Y may be sufficiently small to penetrate a tissue. Y may besufficiently small to penetrate a tumor. Y may comprise a smallmolecule, thus producing a small molecule antibody conjugate (SMAC) whenconjugated to X. The SMAC may be sufficiently small to penetrate atissue. The SMAC may be sufficiently small to penetrate a tumor. Thesmall molecule may be amenable to extensive chemical modification. Thesmall molecule may be derivatized easily with other molecules, includingproteins.

The targeting agent may bind a target cell. The targeting agent may binda cell surface protein or a cell surface marker on a cell. The targetingagent may bind a protein, a peptide, or a biomolecule, wherein theprotein, the peptide or the biomolecule is not bound to a cell. Theprotein, peptide or biomolecule may be circulating in a bloodstream. Theprotein, peptide or biomolecule may be a component of extracellularmatrix. The protein may be an enzyme. The enzyme may have enzymaticactivity. A biomolecule, by non-limiting example, may be selected from afiber, a biopolymer (e.g. collagen), a glycan, a proteoglycan, a lipid,a sterol, a carbohydrate, a nucleic acid and a cellular fragment.

The targeting agent of the targeting agent antibody conjugate may have atherapeutic effect because it brings a cytotoxic effector cell inproximity of a target cell. The therapeutic effect on the intendedindication of the targeting agent antibody construct may be due to thetargeting agent antibody conjugate recruiting a cytotoxic effector cellto the target cell. The therapeutic effect on the intended indication ofthe targeting agent antibody construct may be wholly due to thetargeting agent antibody conjugate recruiting a cytotoxic effector cellto the target cell. The therapeutic effect on the intended indication ofthe targeting agent antibody construct may be predominantly due to thetargeting agent antibody conjugate recruiting a cytotoxic effector cellto the target cell.

The therapeutic effect of the intended indication may be due to thetargeting agent antibody conjugate recruiting a protein, peptide orbiomolecule to the target cell. The therapeutic effect of the intendedindication may wholly due to the targeting agent antibody conjugaterecruiting a protein, peptide or biomolecule to the target cell. Thetherapeutic effect on the intended indication may be at least partiallydue to the targeting agent antibody conjugate recruiting a protein,peptide or biomolecule to the target cell.

The targeting agent alone may not have any therapeutic effect. Thetargeting agent alone may not have any therapeutic effect towards anintended indication of the targeting agent antibody conjugate. Thetargeting agent may not have a therapeutic effect towards the intendedindication of the targeting agent antibody conjugate without beingconjugated to the anti-CD3 antibody or antibody fragment. The dose ofthe therapeutic agent when administered as part of the targeting agentantibody conjugate to provide a therapeutic effect may not have atherapeutic effect when the therapeutic agent is administered alone atthat dose. The targeting agent of the targeting agent antibody conjugatemay not be intended to have any therapeutic effect besides recruitingthe cytotoxic effector cell to the target cell. The targeting agent ofthe targeting agent antibody conjugate may have a therapeutic effect onthe target cell, wherein the therapeutic effect is negligible relativeto the therapeutic effect of recruiting the cytotoxic effector cell,protein, peptide or biomolecule to the target cell. The targeting agentof the targeting agent antibody conjugate may have a therapeutic effecton the target cell, wherein the therapeutic effect is less than thetherapeutic effect of recruiting the cytotoxic effector cell, protein,peptide or biomolecule to the target cell. The binding of the targetingagent to the target cell may induce an unintentional response from thetarget cell. The binding of the targeting agent to the target cell mayinduce an unintentional therapeutic effect in addition to thetherapeutic effect of recruiting the cytotoxic effector cell, protein,peptide or biomolecule to the target cell.

The targeting agent may possess a mass between about 0.1 kDa and about60 kDa. The targeting agent may posses a mass between about 0.1 kDa andabout 55 kDa. The targeting agent may posses a mass between about 0.1kDa and about 50 kDa. The targeting agent may posses a mass betweenabout 0.3 kDa and about 50 kDa. The targeting agent may posses a mass ofabout 0.1 kDa, about 0.2 kDa, about 0.3 kDa, about 0.4 kDa, about 0.5kDa, about 0.6 kDa, about 0.7 kDa, about 0.8 kDa, about 0.9 kDa or about1 kDa. The targeting agent may comprise a mass of about 20 kDa, about 25kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50kDa or about 55 kDa.

The small molecule targeting agent may comprise a prostate specificmembrane antigen (PSMA) inhibitor. PSMA is also known as glutamatecarboxypeptidase II and N-acetyl-L-aspartyl-L-glutamate peptidase I. ThePSMA inhibitor may be 2-[3-(1,3-dicarboxypropy)ureidol]pentanedioic acid(DUPA) or a derivative thereof. The targeting agent antibody conjugatemay comprise an anti-CD3 Fab and two DUPAs, wherein a first of the twoDUPAs is linked by a first linker to the anti-CD3 Fab and a second ofthe two DUPAs is linked by a second linker to the anti-CD3 Fab.

Y may comprise a ligand that binds to at least a portion of a receptoron a cell. Y may comprise a ligand that binds to at least a portion of aco-receptor on a cell. Y may comprise a ligand that binds to at least aportion of an antigen or cell surface marker on a cell. The cell may bea hematopoietic cell. The hematopoietic cell may be a myeloid cell. Themyeloid cell may be an erythrocyte, thrombocyte, neutrophil, monocyte,macrophage, eosinophil, basophil, or mast cell. The hematopoietic cellmay be a lymphoid cell. The lymphoid cell may be a B-cell, T-cell, orNK-cell. The hematopoietic cell may be a leukocyte. The hematopoieticcell may be a lymphocyte. The cell may be a prostate cell. The cell maybe a breast cell. The cell may be a liver cell, kidney cell, lung cell,cardiac cell, muscle cell, nerve cell, neuron, brain cell, epithelialcell, esophageal cell. The cell may be a tumor cell. The cell may be anon-tumor cell. The cell may be an inflammatory cell. The inflammatorycell may be a macrophage. The macrophage may be pro-inflammatory. Themacrophage may be anti-inflammatory. The cell may produce a cytokine.The cell may produce a chemokine.

The cell may be a cancer cell. The cancer cell may be derived from aprosate gland, a breast, an ovary, a cervix, a lung, a kidney, a colon,a rectum, a brain, a thyroid gland, a pancreas, a gastrointestinal tractor a stomach. The cancer cell may be derived from an epithelial tissue,a stromal tissue or an endometrial tissue.

Y may comprise a ligand that binds to a receptor on a cell. The receptormay be a G-protein coupled receptor (GPCR), a tyrosine kinase receptor,a cytokine receptor or an integrin. The receptor may be a growth factorreceptor. The growth factor receptor may be an epidermal growth factorreceptor (EGFR), platelet derived growth factor receptor (PDGFR) orfibroblast growth factor receptor (FGFR). The EGFR may be EGFR1. TheEGFR may be Her2. The receptor may be a cholecystokinin B receptor. Thereceptor may be a gonadotropin-releasing hormone receptor. The receptormay be a somatostatin receptor. The receptor may be somatostatinreceptor 2. The receptor may be a gastrin-releasing peptide receptor.The receptor may be a neurokinin receptor. The may be neurokinin 1receptor, also known as tachykinin 1 receptor. The receptor may be amelanocortin receptor. The receptor may be melanocortin 1 receptor. Thereceptor may be a neurotensin receptor. The receptor may be aneuropeptide Y receptor. The neuropeptide Y receptor (NPYR) may beselected from NPY1R, NPY2R, PPYR1 and NPY5R.

Y may bind an antigen or cell surface marker on a cell. The antigen orcell surface marker may comprise cluster of differentiation protein. Thedifferentiation protein may comprise CD38.

Y may bind an adhesion molecule. The adhesion molecule may be CLL-1.

Y may bind an integrin. The integrin may be an av integrin. The integrinmay be avB3 integrin.

Y may bind a prostate specific membrane antigen (PSMA). Y may compriseDUPA. Y may consist essentially of DUPA.

Y may be a G protein coupled receptor agonist. Y may be a G proteincoupled receptor antagonist. Y may be a cholecystokinin (CCK) receptoragonist. Y may be a cholecystokinin (CCK) receptor antagonist. Y may bea cholecystokinin A receptor agonist. Y may be a cholecystokinin Areceptor antagonist. Y may be a cholecystokinin B receptor agonist. Ymay be a cholecystokinin B receptor antagonist. The cholecystokinin Breceptor antagonist may be pentagastrin.

Y may comprise a hormone. Y may comprise a hormonal ligand. Y maycomprise a hormone receptor agonist. Y may comprise a hormone receptorantagonist. Y may comprise gonadotropin releasing hormone. Y may be amelanocortin receptor ligand. Y may comprise a-melanocyte-stimulatinghormone, afamelanotide, BMS-470,539, bremelanotide, melanotan II oragouti signaling peptide.

Y comprise a neuropeptide. Y may comprise a tachykinin. Y may comprise aneurokinin receptor ligand. Y may comprise substance P. Y may comprse aneurokinin. The neurokinin may be neurokinin A. The neuropeptide maycomprise neuropeptide Y. Y may comprise a neuropeptide Y receptoragonist or a neuropeptide Y receptor antagonist. The neuropeptide Yreceptor agonist may be selected from peptide YY and pancreaticpolypeptide. The neuropeptide Y receptor antagonist may be selected fromBIBP-3226, Lu AA-33810, BIIE-0246 and UR-AK49. The neuropeptide maycomprise a neurotensin receptor ligand. The neurotensin receptor ligandmay be a neurotensin receptor agonist. The neurotensin receptor agonistmay be selected from beta-lactotensin, JMV-449, neurotensin, neuromedinN, PD-149,163 and non-peptide partial agonists derived from SR-48692.The neurotensin receptor ligand may comprise a neurotensin receptorantagonist. The neurotensin receptor antagonist may be selected fromLevocabastine (NTS₂ selective, also H₁ histamine antagonist), SR-48692(NTS₁ selective), or SR-142,948.

Y may comprise a peptide hormone. The peptide hormone may be asomatostatin, or growth hormone inhibiting hormone or somatotropinrelease-inhibiting factor or somatotropin release-inhibiting hormone. Ymay comprise a somatostatin analog. Y may comprise octreotride. Y maycomprise octreotate. The octreotate may be DOTA labeled for ⁶⁸Ga PETimaging and ¹⁷⁷Lu radio therapy.

Y may comprise a peptide. Y may comprise a small peptide. Y may comprisea small peptide ligand. Y may comprise a cyclic peptide. Y may comprisean Arginine-Glycine-Aspartatic Acid (RGD) sequence. Y may comprise anRGD-containing ligand for an integrin. Y may comprise cRGD. Y maycomprise cilengitide. The small peptide may comprise a ligand for agastrin-releasing peptide receptor. The small peptide may comprise aligand for a bombesin receptor selected from BBR1, BBR2 and BBR3. Theligand for a gastrin-releasing peptide receptor may comprise bombesin,neuromedin B, gastrin-releasing peptide. The ligand for thegastrin-releasing peptide receptor may comprise bombesin.

The targeting agent antibody conjugate may comprise a radiolabeledisotope. The targeting agent antibody conjugate may comprise a chelatingagent for a radiolabeled isotope. The targeting agent may comprise aradiolabeled isotope. The targeting agent may comprise a chelating agentfor a radiolabeled isotope. Y may comprise a chelating agent for aradiolabeled isotope. Y may comprise a(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) DOTA. Theradiolabeled isotope may comprise Yttrium. The radiolabeled isotope maybe ⁹⁰Y. DOTA may comprise DOTATOC. DOTA may comprise DOTA-TATE.

Y may comprise at least a portion of a sequence selected from SEQ IDNOs: 3-40. Y may comprise a sequence that is at least 50% identical to asequence selected from SEQ ID NOs: 3-40. Y may comprise a sequence thatis at least 60% identical to a sequence selected from SEQ ID NOs: 3-40.Y may comprise a sequence that is at least 70% identical to a sequenceselected from SEQ ID NOs: 3-40. Y may comprise a sequence that is atleast 80% identical to a sequence selected from SEQ ID NOs: 3-40. Y maycomprise a sequence that is at least 50% identical to a sequenceselected from SEQ ID NOs: 3-40. Y may comprise a sequence that is atleast 85% identical to a sequence selected from SEQ ID NOs: 3-40. Y maycomprise a sequence that is at least 90% identical to a sequenceselected from SEQ ID NOs: 3-40. Y may comprise a sequence that is atleast 95% identical to a sequence selected from SEQ ID NOs: 3-40. Y maycomprise a sequence that is at least 97% identical to a sequenceselected from SEQ ID NOs: 3-40.

Y may comprise a sequence comprising five or more amino acids based onor derived from a sequence selected from SEQ ID NOs: 3-40. Y maycomprise a sequence comprising 6, 7, 8, 9, 10 or more amino acids basedon or derived from a sequence selected from SEQ ID NOs: 3-40. Y maycomprise a sequence comprising 15, 16, 17, 18, 19, 20 or more aminoacids based on or derived from a sequence selected from SEQ ID NOs:3-40. Y may comprise a sequence comprising 25, 30, 35, 40, 45, 50 ormore amino acids based on or derived from a sequence selected from SEQID NOs: 3-40. Y may comprise a sequence comprising 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or more amino acids based on or derived from asequence selected from SEQ ID NOs: 3-40. The amino acids may beconsecutive. The amino acids may be non-consecutive.

Y may comprise a peptide sequence that differs from a peptide sequencebased on or derived from one or more targeting agents by less than orequal to about 20, 17, 15, 12, 10, 8, 6, 5, 4 or fewer amino acids. Ymay comprise a peptide sequence that differs from a peptide sequencebased on or derived from one or more targeting agents by less than orequal to about 4 or fewer amino acids. Y may comprise a peptide sequencethat differs from a peptide sequence based on or derived from one ormore targeting agents by less than or equal to about 3 or fewer aminoacids. Y may comprise a peptide sequence that differs from a peptidesequence based on or derived from one or more targeting agents by lessthan or equal to about 2 or fewer amino acids. Y may comprise a peptidesequence that differs from a peptide sequence based on or derived fromone or more targeting agents by less than or equal to about 1 or feweramino acids. The amino acids may be consecutive, nonconsecutive, or acombination thereof. For example, Y may comprise a peptide sequence thatdiffers from a peptide sequence based on or derived from one or moretargeting agents by less than about 3 consecutive amino acids.Alternatively, or additionally, Y may comprise a peptide sequence thatdiffers from a peptide sequence based on or derived from one or moretargeting agents by less than about 2 non-consecutive amino acids. Inanother example, Y may comprise a peptide sequence that differs from apeptide sequence based on or derived from one or more targeting agentsby less than about 5 amino acids, wherein 2 of the amino acids areconsecutive and 2 of the amino acids are non-consecutive.

Y may comprise a protein or peptide based on a nucleotide sequence thatdiffers from a nucleotide sequence based on or derived from one or moretargeting agents by less than or equal to about 30, 25, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or fewer nucleotides orbase pairs. Y may comprise a protein or peptide based on a nucleotidesequence that differs from a nucleotide sequence based on or derivedfrom one or more targeting agents by less than or equal to about 15 orfewer nucleotides or base pairs. Y may comprise protein or peptide basedon a a nucleotide sequence that differs from a nucleotide sequence basedon or derived from one or more targeting agents by less than or equal toabout 12 or fewer nucleotides or base pairs. Y may comprise a protein orpeptide based on a nucleotide sequence that differs from a nucleotidesequence based on or derived from one or more targeting agents by lessthan or equal to about 9 or fewer nucleotides or base pairs. Y maycomprise a protein or peptide based on a nucleotide sequence thatdiffers from a nucleotide sequence based on or derived from one or moretargeting agents by less than or equal to about 6 or fewer nucleotidesor base pairs. Y may comprise a protein or peptide based on a nucleotidesequence that differs from a nucleotide sequence based on or derivedfrom one or more targeting agents by less than or equal to about 4 orfewer nucleotides or base pairs. Y may comprise a protein or peptidebased on a nucleotide sequence that differs from a nucleotide sequencebased on or derived from one or more targeting agents by less than orequal to about 3 or fewer nucleotides or base pairs. Y may comprise aprotein or peptide based on a nucleotide sequence that differs from anucleotide sequence based on or derived from one or more targetingagents by less than or equal to about 2 or fewer nucleotides or basepairs. Y may comprise a protein or peptide based on a nucleotidesequence that differs from a nucleotide sequence based on or derivedfrom one or more targeting agents by less than or equal to about 1 orfewer nucleotides or base pairs. The nucleotides or base pairs may beconsecutive, nonconsecutive, or a combination thereof. For example, Ymay comprise a protein or peptide based on a nucleotide sequence thatdiffers from a nucleotide sequence based on or derived from one or moretargeting agents by less than about 3 consecutive nucleotides or basepairs. Alternatively, or additionally, Y may comprise a protein orpeptide based on a nucleotide sequence that differs from a nucleotidesequence based on or derived from one or more targeting agents by lessthan about 2 non-consecutive nucleotides or base pairs. In anotherexample, Y may comprise a protein or peptide based on a nucleotidesequence that differs from a nucleotide sequence based on or derivedfrom one or more targeting agents by less than about 5 nucleotides orbase pairs, wherein 2 of the nucleotides or base pairs are consecutiveand 2 of the nucleotides or base pairs are non-consecutive.

The peptide sequence of Y may differ from the peptide sequence of thetargeting agents that it is based on and/or derived from by one or moreamino acid substitutions. The peptide sequence of Y may differ from thepeptide sequence of the targeting agents that it is based on and/orderived from by two or more amino acid substitutions. The peptidesequence of Y may differ from the peptide sequence of the targetingagents that it is based on and/or derived from by three or more aminoacid substitutions. The peptide sequence of Y may differ from thepeptide sequence of the targeting agents that it is based on and/orderived from by four or more amino acid substitutions. The peptidesequence of Y may differ from the peptide sequence of the targetingagents that it is based on and/or derived from by five or more aminoacid substitutions. The peptide sequence of Y may differ from thepeptide sequence of the targeting agents that it is based on and/orderived from by six or more amino acid substitutions. The peptidesequence of Y may differ from the peptide sequence of the targetingagents that it is based on and/or derived from by 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 15, 17, 20, 25 or more amino acid substitutions.

The nucleotide sequence of Y may differ from the nucleotide sequence ofthe targeting agent that it is based on and/or derived from by one ormore nucleotide and/or base pair substitutions. The nucleotide sequenceof Y may differ from the nucleotide sequence of the targeting agent thatit is based on and/or derived from by two or more nucleotide and/or basepair substitutions. The nucleotide sequence of Y may differ from thenucleotide sequence of the targeting agent that it is based on and/orderived from by three or more nucleotide and/or base pair substitutions.The nucleotide sequence of Y may differ from the nucleotide sequence ofthe targeting agent that it is based on and/or derived from by four ormore nucleotide and/or base pair substitutions. The nucleotide sequenceof Y may differ from the nucleotide sequence of the targeting agent thatit is based on and/or derived from by five or more nucleotide and/orbase pair substitutions. The nucleotide sequence of Y may differ fromthe nucleotide sequence of the targeting agent that it is based onand/or derived from by six or more nucleotide and/or base pairsubstitutions. The nucleotide sequence of Y may differ from thenucleotide sequence of the targeting agent that it is based on and/orderived from by nine or more nucleotide and/or base pair substitutions.The nucleotide sequence of Y may differ from the nucleotide sequence ofthe targeting agent that it is based on and/or derived from by twelve ormore nucleotide and/or base pair substitutions. The nucleotide sequenceof Y may differ from the nucleotide sequence of the targeting agent thatit is based on and/or derived from by fifteen or more nucleotide and/orbase pair substitutions. The nucleotide sequence of Y may differ fromthe nucleotide sequence of the targeting agent that it is based onand/or derived from by eighteen or more nucleotide and/or base pairsubstitutions. The nucleotide sequence of Y may differ from thenucleotide sequence of the targeting agent that it is based on and/orderived from by 20, 22, 24, 25, 27, 30 or more nucleotide and/or basepair substitutions.

Y may comprise one or more unnatural amino acids. Y may comprise two ormore unnatural amino acids. Y may comprise three or more unnatural aminoacids. Y may comprise four or more unnatural amino acids. Y may comprise5, 6, 7, 8, 9, 10 or more unnatural amino acids.

Y may be coupled to one or more linkers. Y may be linked to X by one ormore linkers. Y may be linked to X by two or more linkers. Y may belinked to X by three or more linkers.

The distance between X and Y may be between about 1 angstroms (Å) toabout 120 angstroms (Å). The distance between X and Y may be betweenabout 5 angstroms (Å) to about 105 angstroms (Å). The distance between Xand Y may be between about 10 angstroms (Å) to about 100 angstroms (Å).The distance between X and Y may be between about 10 angstroms (Å) toabout 90 angstroms (Å). The distance between X and Y may be betweenabout 10 angstroms (Å) to about 80 angstroms (Å). The distance between Xand Y may be between about 10 angstroms (Å) to about 70 angstroms (Å).The distance between X and Y may be between about 15 angstroms (Å) toabout 45 angstroms (Å). The distance between X and Y may be equal to orgreater than about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 27, 30 or more angstroms. The distance between X and Y maybe equal to or greater than about 10 angstroms. The distance between Xand Y may be equal to or greater than about 15 angstroms. The distancebetween X and Y may be equal to or greater than about 20 angstroms. Thedistance between X and Y may be equal to or less than about 110, 100,90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36,35, 34, 33, 32, 31, 30 or fewer angstroms. The distance between X and Ymay be equal to or less than about 100 angstroms. The distance between Xand Y may be equal to or less than about 80 angstroms. The distancebetween X and Y may be equal to or less than about 60 angstroms. Thedistance between X and Y may be equal to or less than about 40angstroms.

II. Linkers

The targeting agent antibody conjugates disclosed herein may compriseone or more linkers (e.g., L1, L2). The targeting agent antibodyconjugates disclosed herein may comprise two or more linkers. Thetargeting agent antibody conjugates disclosed herein may comprise threeor more linkers. The targeting agent antibody conjugates disclosedherein may comprise 4, 5, 6, 7 or more linkers.

The linker may comprise a chemical bond. The linker may comprise afunctional group. The linker may comprise an amino acid. The linker maycomprise a peptide. The linker may comprise a polymer. The polymer maybe a polyethylene glycol.

The one or more linkers may comprise one or more reactive functionalgroups that may react with a complementary reactive functional group ona coupling partner. The linker may be bifunctional. The bifunctionallinker may be heterobifunctional. The linker may comprise ethyleneglycol. The linker may be a bifunctional ethylene glycol linker.

One or more linkers may be formed by reaction of an amino acid on X witha linker already attached to Y. One or more linkers may be formed byreaction of an amino acid or another reactive functional group on Y witha linker already attached to X. One or more linkers may be formed byreaction of a linker already attached to X with another linker alreadyattached to Y. In order to form a linker already attached to X or Y, abifunctional linker, with two orthogonally reactive functional groups,may be coupled to X or Y, such that one remaining reactive functionalgroup is available for subsequent coupling.

The linker may be the product of a bioorthogonal reaction, non-limitingexamples of which are reviewed in Kim et al., Curr Opin Chem Bio17:412-419 (2013). The linker may comprise an oxime, a tetrazole, aDiels Alder adduct, a hetero Diels Alder adduct, an aromaticsubstitution reaction product, a nucleophilic substitution reactionproduct, an ester, an amide, a carbamate, an ether, a thioether, or aMichael reaction product. The linker may be a cycloaddition product, ametathesis reaction product, a metal-mediated cross-coupling reactionproduct, a radical polymerization product, an oxidative couplingproduct, an acyl-transfer reaction product, or a photo click reactionproduct. The cycloaddition may be a Huisgen-cycloaddition. Thecycloaddition may be a copper-free [3+2] Huisgen-cycloaddition. Thecycloaddition may be a Diels-Alder reaction. The cycloaddition may be ahetero Diels-Alder reaction. The linker may be the product of anenzyme-mediated reaction. The linker may be a product of atransglutaminase-mediated reaction, non-limiting examples of which aredescribed in Lin et al., J. Am. Chem. Soc. 128:4542-4543 (2006) and WO2013/093809. The linker may comprise a disulfide bridge that connectstwo cysteine residues, such as ThioBridge™ technology by PolyTherics.The linker may comprise a maleimide bridge that connects two amino acidresidues. The linker may comprise a maleimide bridge that connects twocysteine residues.

Each of the one or more linkers may comprise one or more ethyleneglycols. Each of the one or more linkers may comprise at least onereactive functional group selected from alkoxy-amine, hydrazine,aryl/alkyl azide, alkyne, alkene, tetrazine, dichlorotriazine,tresylate, succinimidyl carbonate, benzotriazole carbonate, nitrophenylcarbonate, trichlorophenyl carbonate, carbonylimidazole, succinimidylsuccinate, maleimide, vinylsulfone, haloacetamide, and disulfide. Thealkene may be selected from norbornene, trans-cyclooctene, andcyclopropene. Each of the one or more linkers may comprise at least onealkoxy amine. Each of the one or more linkers may comprise at least oneazide. Each of the one or more linkers may comprise at least onecyclooctyne. Each of the one or more linkers may comprise at least onetetrazine.

The linker may couple with one or more natural amino acids on X or Y.The linker may couple with one or more unnatural amino acids on X or Y.The linker may couple with an amino acid which is the product ofsite-specific mutagenesis. The linker may couple with a cysteine whichis the product of site-specific mutagenesis. The linker (e.g.,substituted maleimide) may couple with a cysteine which is the productof site-specific mutagenesis, as well as a native cysteine residue. Twolinkers, each with complementary reactive functional groups, may couplewith one another.

The one or more linkers may comprise a cleavable linker. The one or morelinkers may comprise a non-cleavable linker. The one or more linkers maycomprise a flexible linker. The one or more linkers may comprise aninflexible linker.

The ethylene glycol linker may comprise about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19 or about 20 ethylene glycol subunits. The one or more linkersmay cmoprise a 1,4-dicarboxylic moiety. The one or more linkers maycomprise a 1,3-dinitro substituted phenyl moiety. The one or morelinkers may comprise

The one or more linkers may comprise an alkoxy-amine (or aminooxy)group, azide group and/or cyclooctyne group at one or more termini. Theone or more linkers may comprise an alkoxy-amine at one terminus and anazide group at the other terminus. The one or more linkers may comprisean alkoxy-amine at one terminus and a cyclooctyne group at the otherterminus. The alkoxy-amine may form a stable oxime with a ketone groupon an amino acid. The alkoxy-amine may form a stable oxime with a ketonegroup on an unnatural amino acid. The ketone group may be on ap-acetylphenylalanine (pAcF).

The one or more linkers may be coupled to X, Y, or a combinationthereof. The one or more linkers may be coupled to X and/or Y to formone or more intermediates of the Formula III: L1-X, Formula IIIA: X-L1,Formula IV: L1-Y or Formula IVA: Y-L1. The one or more linkers may becoupled to X and/or Y by an oxime. The one or more linkers may becoupled to X and/or Y by a cyclooctyne, cyclopropene, arly/alkyl azides,trans-cyclooctene, norborene, tetrazine, or a combination thereof. Theone or more linkers may be coupled to X and/or Y by a covalent bond,non-covalent bond, ionic bond, or a combination thereof.

The two or more linkers may be linked. The two or more linkers may belinked through one or more copper-free reactions. The two or morelinkers may be linked through one or more cycloadditions. The two ormore linkers may be linked through one or more Huisgen-cycloadditions.The two or more linkers may be linked through one or more copper-free[3+2] Huisgen-cycloadditions. The two or more linkers may be linkedthrough one or more copper-containing reactions. The two or more linkersmay be linked through one or more Diels Alder reactions. The two or morelinkers may be linked through one or more hetero Diels Alder reactions.

Targeting agent antibody conjugates may be optimized by adjusting linkerlength. Linkers may be relatively short. Linkers may be relatively long.The one or more linkers may be between about 1 angstroms (Å) to about120 angstroms (Å) in length. The one or more linkers may be betweenabout 5 angstroms (Å) to about 105 angstroms (Å) in length. The one ormore linkers may be between about 10 angstroms (Å) to about 100angstroms (Å) in length. The one or more linkers may be between about 10angstroms (Å) to about 90 angstroms (Å) in length. The one or morelinkers may be between about 10 angstroms (Å) to about 80 angstroms (Å)in length. The one or more linkers may be between about 10 angstroms (Å)to about 70 angstroms (Å) in length. The one or more linkers may bebetween about 15 angstroms (Å) to about 45 angstroms (Å) in length. Theone or more linkers may be equal to or greater than about 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or moreangstroms in length. The one or more linkers may be equal to or greaterthan about 10 angstroms in length. The one or more linkers may be equalto or greater than about 15 angstroms in length. The one or more linkersmay be equal to or greater than about 20 angstroms in length. The one ormore linkers may be equal to or less than about 110, 100, 90, 85, 80,75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33,32, 31, 30 or fewer angstroms in length. The one or more linkers may beequal to or less than about 100 angstroms in length. The one or morelinkers may be equal to or less than about 80 angstroms in length. Theone or more linkers may be equal to or less than about 60 angstroms inlength. The one or more linkers may be equal to or less than about 40angstroms in length.

The total length of the linkers may be between about 1 angstroms (Å) toabout 120 angstroms (Å). The total length of the linkers may be betweenabout 5 angstroms (Å) to about 105 angstroms (Å). The total length ofthe linkers may be between about 10 angstroms (Å) to about 100 angstroms(Å). The total length of the linkers may be between about 10 angstroms(Å) to about 90 angstroms (Å). The total length of the linkers may bebetween about 10 angstroms (Å) to about 80 angstroms (Å). The totallength of the linkers may be between about 10 angstroms (Å) to about 70angstroms (Å). The total length of the linkers may be between about 15angstroms (Å) to about 45 angstroms (Å). The total length of the linkersmay be equal to or greater than about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or more angstroms. The totallength of the linkers may be equal to or greater than about 10angstroms. The total length of the linkers may be equal to or greaterthan about 15 angstroms. The total length of the linkers may be equal toor greater than about 20 angstroms. The total length of the linkers maybe equal to or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55,50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30 or fewerangstroms. The total length of the linkers may be equal to or less thanabout 100 angstroms. The total length of the linkers may be equal to orless than about 80 angstroms. The total length of the linkers may beequal to or less than about 60 angstroms. The total length of thelinkers may be equal to or less than about 40 angstroms. ProstateSpecific Membrane Antigen Binding Small Molecule Antibody Conjugate(PSMA C)

The targeting agent antibody conjugate may comprise an anti-CD3 Fab; oneor more DUPA molecules; and one or more linkers, wherein the antibody orantibody fragment is linked to the one or more targeting agents by theone or more linkers. The anti-CD3 Fab may comprise one or more unnaturalamino acids. A first unnatural amino acid and a second unnatural aminoacid may replace a natural amino acid of the anti-CD3 Fab. The firstDUPA molecule and the second DUPA molecule may be site-specificallylinked to a first unnatural amino acid and a second unnatural amino acidof the anti-CD3 Fab. The natural amino acid may be selected from alanine(Ala), lysine (Lys), serine (Ser) and/or threonine (Thr) residue of Xand/or Y. The natural amino acid that is replaced may be selected fromLysine 138 (Lys 138) of a heavy chain of the anti-CD3 Fab, Alanine 123(Ala 123) of a heavy chain of the anti-CD3 Fab, Threonine 109 (Thr 109)of a heavy chain of the anti-CD3 Fab and Serine 202 (Ser 202) of a heavychain of the anti-CD3 Fab. The targeting agent antibody conjugate may beof Formula I: X-L1-Y or Formula IA: Y-L1-X, wherein: X comprises theanti-CD3 Fab; L1 comprises the one or more linkers; and Y comprises oneor more DUPA molecules. The targeting agent antibody conjugate maycomprise a compound selected from the compounds of Formula V, FormulaVI, Formula VII and Formula VIII:

The targeting agent antibody conjugate may comprise a compound ofFormula IX:

The targeting agent antibody conjugate may comprise a compound ofFormula X:

The targeting agent antibody conjugate may comprise a compound ofFormula XI:

In another aspect, provided herein are compounds of Formula XII:

Y-L-A¹-L¹-A²-L²-A³-L³-X²  (Formula XII)

wherein:

-   -   Y is a ligand of prostate specific membrane antigen (PSMA);    -   L is

-   -   A¹ is selected from the group consisting of an aryl, a 5- to        6-membered heteroaryl, —C(O)—, —N(R¹)—, —O—, —C(O)N(R¹)—,        —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—, and —N(R¹)S(O)_(1,2)—;    -   L¹ is

-   -   A² is selected from the group consisting of a bond, —C(O)—,        —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—, and        —N(R¹)S(O)_(1,2)—;    -   L² is

-   -   A³ is a bond,

-   -   L³ is

-   -   X² is a linker bound to a functional group that reacts with an        amino acid, or a linker bound to a modified amino acid, wherein        the modified amino acid is part of X, wherein X is a modified        therapeutic peptide, protein, or antibody;    -   each R¹ is independently selected from H, alkyl, or haloalkyl;    -   each R², R²¹, R²², and R²³ is independently selected from H,        halo, —OR¹, —CN, —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl,        or heteroarylalkyl;    -   each R³ is independently selected from halo, —OR¹, —CN, —SR¹,        alkyl, cycloalkyl, haloalkyl, arylalkyl, or heteroarylalkyl,        —NO₂, and NR¹R¹;    -   each G¹ and G² is independently selected from the group        consisting of a bond, —C(O)—, —N(R¹)—, —O—, —C(O)N(R¹)—,        —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—, and —N(R¹)S(O)_(1,2)—;    -   each Z, Z¹, Z², and Z³ is independently selected from the group        consisting of a bond, —O—, and —N(R¹)—;    -   k, k¹, k² and k³ are each independently selected from 0, 1, 2,        3, 4, 5, 6, 7, 8, 9, and 10;    -   m¹, m² and m³ are each independently selected from 0, 1, 2, 3,        4, 5, 6, 7, 8, 9, and 10; and    -   p is 0, 1, 2, 3 or 4;    -   or a stereoisomer thereof.

In some embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIa:

wherein:

-   -   Q is selected from the group consisting of:

-   -    and    -   E is selected from the group consisting of:

In some embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIb:

In further embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIc:

In some embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIId:

In further embodiments described above or below of a compound of FormulaXII, the compound is of Formula XIIe:

In still further embodiments described above or below of a compound ofFormula XII, the compound is of Formula XIIf:

In some embodiments described above or below of a compound of FormulaXII, A¹ is —C(O)N(H)—. In some embodiments described above or below of acompound of Formula XII, A¹ is

In some embodiments described above or below of a compound of FormulaXII, A³ is

In further embodiments described above or below of a compound of FormulaXII, A³ is

In some embodiments described above or below of a compound of FormulaXII, each R², R²¹, R²², R²³, and R²⁴ is independently selected from H,F, —CH₃, or —CF₃. In some embodiments described above or below of acompound of Formula XII, each R², R²¹, R²², R²³, and R²⁴ is H.

In some embodiments described above or below of a compound of FormulaXII,

-   -   X² is

-   -   wherein:        -   A⁴ is selected from the group consisting of a bond, —C(O)—,            —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)_(1,2)N(R¹)—,            and —N(R¹)S(O)_(1,2)—;        -   each R²⁴ is independently selected from H, halo, —OR¹, —CN,            —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl, or            heteroarylalkyl;        -   k⁴ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;        -   Z⁴ is selected from a bond, aryl, and a 5- to 6-membered            heteroaryl; and        -   X¹ is —ONH₂,

-   -   -    —N₃,

-   -   -    —N(H)NH₂, or —SH.

In some embodiments described above or below of a compound of FormulaXII, X² is

In further embodiments described above or below of a compound of FormulaXII, X² is

In some embodiments described above or below of a compound of FormulaXII, the compound is selected from:

or a stereoisomer thereof.

In some embodiments described above or below of a compound of FormulaXII,

-   -   X² is

-   -   wherein:        -   A⁴ is selected from the group consisting of a bond, —C(O)—,            —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)₁₂N(R¹)—, and            —N(R¹)S(O)_(1,2)—;        -   each R²⁴ is independently selected from H, halo, —OR¹, —CN,            —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl, or            heteroarylalkyl;        -   k⁴ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;        -   Z⁴ is selected from a bond, aryl, and a 5- to 6-membered            heteroaryl; and

-   -   -    H or —S—;        -   X is a modified therapeutic peptide, protein, or antibody;        -   L⁴ is a bond directly attached to a modified amino acid, or            a linker bound to a modified amino acid, wherein the            modified amino acid is part of X.            In some embodiments described above or below of a compound            of Formula XII, the amino acid is an unnatural amino acid.

In some embodiments described above or below of a compound of FormulaXII, k is 1, 2, or 3; and Z is a bond.

In some embodiments described above or below of a compound of FormulaXII, A¹ is —C(O)N(R¹)—, 6-membered aryl, or 5-membered heteroaryl.

In some embodiments described above or below of a compound of FormulaXII, m¹ is 0; k¹ is 6 or 7; and Z¹ is a bond.

In some embodiments described above or below of a compound of FormulaXII, A² is a bond; m² and k² are 0; and Z² is a bond.

In some embodiments described above or below of a compound of FormulaXII, A² is —C(O)N(H)—; m² is 2; k² is 2; and Z² is a bond. In someembodiments described above or below of a compound of Formula XII, A² is—C(O)N(H)—; m² is 3; k² is 2; and Z² is a bond. In some embodimentsdescribed above or below of a compound of Formula XII, A² is —C(O)N(H)—;m² is 10; k² is 2; and Z² is a bond.

In some embodiments described above or below of a compound of FormulaXII, R³ is —NO₂; and p is 2.

In some embodiments described above or below of a compound of FormulaXII, each GX¹ and GX² are independently selected from the groupconsisting of —N(H)—, —C(O)N(H)—, and —N(H)C(O)—.

In some embodiments described above or below of a compound of FormulaXII, m³ is 3; k³ is 2; and Z³ is a bond. In some embodiments describedabove or below of a compound of Formula XII, m³ is 2; k³ is 2; and Z³ isa bond.

In some embodiments described above or below of a compound of FormulaXII, A³ is a bond; m³ and k³ are 0; and Z³ is a bond.

III. Unnatural Amino Acids

The targeting agent antibody conjugates disclosed herein may compriseone or more unnatural amino acids. As used herein, the terms “unnaturalamino acid” and “non-natural amino acid” refer to non-proteinogenicamino acids that either occur naturally or are chemically synthesized.The targeting agent antibody conjugates disclosed herein may comprise X,wherein X comprises one or more unnatural amino acids. The targetingagent antibody conjugates disclosed herein may comprise Y, wherein Ycomprises one or more unnatural amino acids. The antibodies disclosedherein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more unnatural aminoacids. The unnatural amino acid may react with the linker to create achemical bond.

The one or more unnatural amino acids may be incorporated into anantibody (e.g., antibody of Formula I, IA, II, and IIA); intermediate(e.g., intermediate of Formula III, IIIA, IV, IVA); X antibody orantibody fragment; Y (e.g. peptide); or a combination thereof. The oneor more unnatural amino acids may be site-specifically incorporated intoan antibody (e.g., antibody of Formula I, IA, II, and IIA); intermediate(e.g., intermediate of Formula III, IIIA, IV, IVA); X antibody orantibody fragment; Y peptide; or a combination thereof. The one or moreunnatural amino acids may be incorporated into an antibody fragment ofan antibody (e.g., antibody of Formula I, IA, II, and IIA); intermediate(e.g., intermediate of Formula III, IIIA, IV, IVA); X antibody orantibody fragment; Y peptide; or a combination thereof.

The one or more unnatural amino acids may be incorporated into a heavychain, light chain, variable region, constant region, Fab fragment of anantibody (e.g., antibody of Formula I, IA, II, and IIA); intermediate(e.g., intermediate of Formula III, IIIA, IV, IVA); X antibody orantibody fragment; Y peptide; or a combination thereof.

The one or more unnatural amino acids may be inserted between twonaturally occurring amino acids in the antibody or antibody fragment.The one or more unnatural amino acids may replace one or more naturallyoccurring amino acids in the antibody or antibody fragment. The one ormore unnatural amino acids may be incorporated at the N terminus of theantibody or antibody fragment. The one or more unnatural amino acids maybe incorporated at the C terminus of the antibody or antibody fragment.The unnatural amino acid may be incorporated distal to the bindingregion of the antibody or antibody fragment. The unnatural amino acidmay be incorporated near the binding region of the antibody or antibodyfragment. The unnatural amino acid may be incorporated in the bindingregion of the antibody or antibody fragment.

The one or more unnatural amino acids may be inserted between twonaturally occurring amino acids in the targeting agent. The one or moreunnatural amino acids may replace one or more naturally occurring aminoacids in the targeting agent. The one or more unnatural amino acids maybe incorporated at the N terminus of the targeting agent. The one ormore unnatural amino acids may be incorporated at the C terminus of thetargeting agent. The unnatural amino acid may be incorporated distal tothe binding region of the targeting agent. The unnatural amino acid maybe incorporated near the binding region of the targeting agent. Theunnatural amino acid may be incorporated in the binding region of thetargeting agent.

The one or more unnatural amino acids may be encoded by a codon thatdoes not code for one of the twenty natural amino acids. The one or moreunnatural amino acids may be encoded by a nonsense codon (stop codon).The stop codon may be an amber codon. The amber codon may comprise a UAGsequence. The stop codon may be an ochre codon. The ochre codon maycomprise a UAA sequence. The stop codon may be an opal or umber codon.The opal or umber codon may comprise a UGA sequence. The one or moreunnatural amino acids may be encoded by a four-base codon.

The one or more unnatural amino acids may bep-acetylphenylalanine (pAcFor pAcPhe). The one or more unnatural amino acids may be selenocysteine.The one or more unnatural amino cids may be p-fluorophenylalanine(pFPhe). The one or more unnatural amino acids may be selected from thegroup comprising p-azidophenylalanine (pAzF), p-benzoylphenylalanine(pBpF), p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF),p-cyanophenylalanine (pCNF), p-carboxylmethylphenylalanine (pCmF),3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF),o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3-yl)alanine (HQA),selenocysteine, and (2,2′-bipyridin-5-yl)alanine (BipyA).

The one or more unnatural amino acids may be j3-amino acids (j33 and32), homo-amino acids, proline and pyruvic acid derivatives,3-substituted alanine derivatives, glycine derivatives, ring-substitutedphenylalanine and tyrosine derivatives, linear core amino acids, diaminoacids, D-amino acids, N-methyl amino acids, or a combination thereof.

Additional examples of unnatural amino acids include, but are notlimited to, 1) various substituted tyrosine and phenylalanine analoguessuch as O-methyl-L-tyrosine, p-amino-L-phenylalanine,3-nitro-L-tyrosine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanineand p-isopropyl-L-phenylalanine; 2) amino acids with aryl azide andbenzophenone groups that may be photo-cross-linked; 3) amino acids thathave unique chemical reactivity including acetyl-L-phenylalanine andm-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-(2-propynyl)-L-tyrosine,p-ethylthiocarbonyl-L-phenylalanine andp-(3-oxobutanoyl)-L-phenylalanine; 4) heavy-atom-containing amino acidsfor phasing in X-ray crystallography including p-iodo andp-bromo-L-phenylalanine; 5) the redox-active amino aciddihydroxy-L-phenylalanine; 6) glycosylated amino acids includingb-N-acetylglucosamine-O-serine and a-N-acetylgalactosamine-O-threonine;7) fluorescent amino acids with naphthyl, dansyl, and 7-aminocoumarinside chains; 8) photocleavable and photoisomerizable amino acids withazobenzene and nitrobenzyl Cys, Ser, and Tyr side chains; 9) thephosphotyrosine mimetic p-carboxymethyl-L-phenylalanine; 10) theglutamine homologue homoglutamine; and 11) 2-aminooctanoic acid. Theunnatural amino acid may be modified to incorporate a chemical group.The unnatural amino acid may be modified to incorporate a ketone group.

The one or more unnatural amino acids may comprise at least one oxime,carbonyl, dicarbonyl, hydroxylamine group or a combination thereof. Theone or more unnatural amino acids may comprise at least one carbonyl,dicarbonyl, alkoxy-amine, hydrazine, acyclic alkene, acyclic alkyne,cyclooctyne, aryl/alkyl azide, norbornene, cyclopropene,trans-cyclooctene, or tetrazine functional group or a combinationthereof.

The one or more unnatural amino acids may be incorporated into X and/orY by methods known in the art. Cell-based or cell-free systems may beused to alter the genetic sequence of X and/or Y, thereby producing Xand/or Y with one or more unnatural amino acids. Auxotrophic strains maybe used in place of engineered tRNA and synthetase. The one or moreunnatural amino acids may be produced through selective reaction of oneor more natural amino acids. The selective reaction may be mediated byone or more enzymes. In one non-limiting example, the selective reactionof one or more cysteines with formylglycine generating enzyme (FGE) mayproduce one or more formylglycines as described in Rabuka et al., NatureProtocols 7:1052-1067 (2012).

The one or more unnatural amino acids may take part in a chemicalreaction to form a linker. The chemical reaction to form the linker maybe a bioorthogonal reaction. The chemical reaction to form the linkermay be click chemistry.

Additional unnatural amino acids are disclosed in Liu et al. (Annu RevBiochem, 79:413-44, 2010), Wang et al. (Angew Chem Int Ed, 44:34-66,2005) and PCT application numbers PCT/US2012/039472, PCT/US2012/039468,PCT/US2007/088009, PCT/US2009/058668, PCT/US2007/089142,PCT/US2007/088011, PCT/US2007/001485, PCT/US2006/049397,PCT/US2006/047822 and PCT/US2006/044682, all of which are incorporatedby reference in their entireties.

IV. Targeting Agent Antibody Conjugate Compositions

Disclosed herein are compositions comprising one or more targeting agentantibody conjugates disclosed herein. The compositions may comprise atargeting agent antibody conjugate comprising (a) an antibody orantibody fragment comprising one or more unnatural amino acids; (b) oneor more linkers; and (c) a targeting agent, wherein the one or morelinkers links the antibody or antibody fragment to the targeting agent.The one or more linkers links the antibody or antibody fragment to thetargeting agent site-specifically. The antibody or antibody fragment maycomprise one or more unnatural amino acids. The targeting agent maycomprise one or more unnatural amino acids. The composition may furthercomprise one or more pharmaceutically acceptable excipients. Thecomposition may further comprise one or more solvents or diluents. Thecomposition may further comprise one or more pharmaceutical carriers.

The composition may comprise a targeting agent antibody conjugate ofFormula I: X-L1-Y, wherein (i) X comprises an antibody or antibodyfragment; (ii) Llcomprises one or more linkers; and (iii) Y comprises atargeting agent. The antibody or antibody fragment and the targetingagent may be site-specifically linked by the one or more linkers. Theantibody, antibody fragment and/or targeting agent may comprise one ormore unnatural amino acids. The antibody or antibody fragment and thetargeting agent may be linked at the site of the one or more unnaturalamino acids. The composition may further comprise one or morepharmaceutically acceptable excipients. The composition may furthercomprise one or more solvents or diluents. The composition may furthercomprise one or more pharmaceutical carriers.

The composition may comprise a targeting agent antibody conjugate ofFormula IA: Y-L1-X, wherein (i) X comprises an antibody or antibodyfragment; (ii) Llcomprises one or more linkers; and (iii) Y comprises atargeting agent. The antibody or antibody fragment and the targetingagent may be site-specifically linked by the one or more linkers. Theantibody, antibody fragment and/or targeting agent may comprise one ormore unnatural amino acids. The antibody or antibody fragment and thetargeting agent may be linked at the site of the one or more unnaturalamino acids. The composition may further comprise one or morepharmaceutically acceptable excipients. The composition may furthercomprise one or more solvents or diluents. The composition may furthercomprise one or more pharmaceutical carriers.

The composition may comprise a targeting agent antibody conjugate ofFormula II: X-L¹-L²-Y, wherein (i) X comprises an antibody or antibodyfragment; (ii) L1 and L2 comprise one or more linkers; and (iii) Ycomprises a targeting agent. The antibody or antibody fragment and thetargeting agent may be site-specifically linked by the one or morelinkers. The antibody, antibody fragment and/or targeting agent maycomprise one or more unnatural amino acids. The antibody or antibodyfragment and the targeting agent may be linked at the site of the one ormore unnatural amino acids. The composition may further comprise one ormore pharmaceutically acceptable excipients. The composition may furthercomprise one or more solvents or diluents. The composition may furthercomprise one or more pharmaceutical carriers.

The composition may comprise a targeting agent antibody conjugate ofFormula IIA: Y-L2-L1-X, wherein (i) X comprises an antibody or antibodyfragment; (ii) Lland L2 comprise one or more linkers; and (iii) Ycomprises a targeting agent. The antibody or antibody fragment and thetargeting agent may be site-specifically linked by the one or morelinkers. The antibody, antibody fragment and/or targeting agent maycomprise one or more unnatural amino acids. The antibody or antibodyfragment and the targeting agent may be linked at the site of the one ormore unnatural amino acids. The composition may further comprise one ormore pharmaceutically acceptable excipients. The composition may furthercomprise one or more solvents or diluents. The composition may furthercomprise one or more pharmaceutical carriers.

The term “pharmaceutically acceptable” as used herein, refers to amaterial that does not abrogate the biological activity or properties ofthe agents described herein, and is relatively nontoxic (i.e., thetoxicity of the material significantly outweighs the benefit of thematerial). In some instances, a pharmaceutically acceptable material maybe administered to an individual without causing significant undesirablebiological effects or significantly interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

Pharmaceutical compositions herein may be formulated using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of the active agents into preparations whichare used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen. A summary of pharmaceutical compositionsis found, for example, in Remington: The Science and Practice ofPharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995);Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; andPharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins, 1999).

A pharmaceutical composition disclosed herein may further comprise apharmaceutically acceptable diluent(s), excipient(s), or carrier(s). Thepharmaceutical compositions may include other medicinal orpharmaceutical agents, carriers, adjuvants, such as preserving,stabilizing, wetting or emulsifying agents, solution promoters, saltsfor regulating the osmotic pressure, and/or buffers. In addition, thepharmaceutical compositions also contain other therapeutically valuablesubstances.

A pharmaceutical composition disclosed herein may be administered to asubject by any suitable administration route, including but not limitedto, parenteral (intravenous, subcutaneous, intraperitoneal,intramuscular, intravascular, intrathecal, intravitreal, infusion, orlocal), topical, oral, or nasal administration. A suitableadministration route may comprise a microneedle device.

Formulations suitable for intramuscular, subcutaneous, peritumoral, orintravenous injection may include physiologically acceptable sterileaqueous or non-aqueous solutions, dispersions, suspensions or emulsions,and sterile powders for reconstitution into sterile inj ectablesolutions or dispersions. Examples of suitable aqueous and non-aqueouscarriers, diluents, solvents, or vehicles including water, ethanol,polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor andthe like), suitable mixtures thereof, vegetable oils (such as olive oil)and injectable organic esters such as ethyl oleate. Proper fluidity ismaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection also contain optional additives such aspreserving, wetting, emulsifying, and dispensing agents.

For intravenous injections, an active agent may be optionally formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer.

Parenteral injections optionally involve bolus injection or continuousinfusion. Formulations for injection are optionally presented in unitdosage form, e.g., in ampoules or in multi dose containers, with anadded preservative. The pharmaceutical composition described herein maybe in a form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of an active agent in water soluble form.Additionally, suspensions are optionally prepared as appropriate oilyinjection suspensions.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation may be divided into unit doses containingappropriate quantities of an active agent disclosed herein. The unitdosage may be in the form of a package containing discrete quantities ofthe formulation. Non-limiting examples are packaged tablets or capsules,and powders in vials or ampoules. Aqueous suspension compositions may bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers are used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection are presented in unit dosageform, which include, but are not limited to ampoules, or in multi dosecontainers, with an added preservative.

The pharmaceutical composition may be administered at a dosage of about0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg,1.4 mg/kg, about 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg,2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg or about 3.0 mg/kg. Thepharmaceutical composition may be administered at a dosage of about 3.5mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg,9.0 mg/kg or about 10 mg/kg.

The pharmaceutical composition may be administered once daily, twicedaily, three times daily or more. The pharmaceutical composition may beadministered once weekly, twice weekly, three times weekly or more. Thepharmaceutical composition may be administered bi-weekly. Thepharmaceutical composition may be administered montly. Thepharmaceutical composition may be administered as needed.

The pharmaceutical composition may be co-administered with a therapeutictreatment. The therapeutic treatment may comprise an anti-inflammatorytreatment. The anti-inflammatory treatment may comprise a steroid. Theanti-inflammatory treatment may comprise a non-steroid. The therapeutictreatment may comprise an antibiotic. The therapeutic treatment maycomprise anti-viral drug. The therapeutic treatment may comprise achemotherapy. The therapeutic treatment may comprise a radiation. Thetherapeutic treatment may comprise a bi-specific antibody. Thetherapeutic treatment may comprise an additional targeting agentantibody conjugate.

V. Targeting AGent Antibody Conjugate Production Methods

Disclosed herein are methods of producing targeting agent antibodyconjugate of Formula I: X-L1-Y or Formula IA: Y-L1-X. The method maycomprise coupling one or more linkers to Y, wherein Y comprises thetargeting agent, to produce an intermediate Y-L1 or L1-Y; andconjugating the intermediate to X, wherein X comprises at least aportion of an antibody or antibody fragment, thereby producing thetargeting agent antibody conjugate. The method may further compriseconjugating the intermediate to X and/or Y site-specifically. The methodmay further comprise incorporating one or more unnatural amino acidsinto X and/or Y. Alternatively, the method may comprise coupling one ormore linkers to the antibody or antibody fragment to produce a anintermediate X-L1 or L1X, and conjugating the intermediate to Y, whereinY comprises a targeting agent, thereby producing the targeting agentantibody conjugate . . . Conjugating the intermediate X-L1 or L1X to thetargeting agent may comprise forming an oxime. Forming an oxime mayrequire acidic conditions. The method may further comprise conjugatingthe intermediate to X and/or Y site-specifically. The method may furthercomprise incorporating one or more unnatural amino acids into X and/orY.

Further disclosed herein is a method of producing the targeting agentantibody conjugate of Formula II: X-L1-L2-Y, comprising (a)incorporating one or more unnatural amino acids into X and/or Y, wherein(i) X comprises at least a portion of an antibody or antibody fragment;and (ii) Y comprises at least a portion of a peptide or protein; (b)coupling L1 to X to produce a first intermediate of Formula III: X-L1and coupling L2 to Y to produce a second intermediate of Formula IV:L2-Y; and (c) linking the first intermediate of Formula III to thesecond intermediate of Formula IV, thereby producing the targeting agentantibody conjugate of Formula II.

The method of producing the targeting agent antibody conjugate mayfurther comprise engineering a nucleic acid encoding the antibody orantibody fragment. The method may further comprise incorporating anon-sense codon into the nucleic acid. The non-sense codon may be anamber codon. The method may further comprise incorporating an unnaturalamino acid into the nucleic acid site-specifically. The method mayfurther comprise incorporating an unnatural amino acid into the nucleicacid at the non-sense codon. The method may further comprise expressingthe nucleic acid in a cell. The method may further compriseco-expressing an orthogonal tRNA/tyrosyl-tRNA synthetase pair. Theorthogonal tRNA/tyrosyl-tRNA synthetase pair may incorporate theunnatural amino acid selectively in response to the non-sense codon.

VA. Incorporation of Unnatural Amino Acids

Incorporating one or more unnatural amino acids into the antibody orantibody fragment may comprise modifying one or more amino acid residuesin the antibody or antibody fragment. Modifying the one or more aminoacid residues in the antibody or antibody fragment may comprise mutatingone or more nucleotides in the nucleotide sequence encoding thetargeting agent. Mutating the one or more nucleotides in the nucleotidesequence encoding the targeting agent may comprise altering a codonencoding an amino acid to a nonsense codon.

Incorporating one or more unnatural amino acids into the antibody orantibody fragment may comprise modifying one or more amino acid residuesin the antibody or antibody fragment to produce one or more amber codonsin the antibody or antibody fragment.

The one or more unnatural amino acids may be incorporated into theantibody or antibody fragment in response to an amber codon. The one ormore unnatural amino acids may be site-specifically incorporated intothe antibody or antibody fragment.

Incorporating one or more unnatural amino acids into the antibody orantibody fragment may comprise use of one or more genetically encodedunnatural amino acids with orthogonal chemical reactivity relative tothe canonical twenty amino acids to site-specifically modify thetargeting agent. Incorporating the one or more unnatural amino acids maycomprise use of an evolved tRNA/aminoacyl-tRNA synthetase pair tosite-specifically incorporate one or more unnatural amino acids atdefined sites in the targeting agent in response to one or more ambernonsense codon.

Incorporating one or more unnatural amino acids into a targeting agentmay comprise modifying one or more amino acid residues in a targetingagent. Modifying the one or more amino acid residues in a targetingagent may comprise mutating one or more nucleotides in the nucleotidesequence encoding the targeting agent. Mutating the one or morenucleotides in the nucleotide sequence encoding the targeting agent maycomprise altering a codon encoding an amino acid to a nonsense codon.

Incorporating one or more unnatural amino acids into a targeting agentmay comprise modifying one or more amino acid residues in a targetingagent to produce one or more amber codons in a targeting agent.

The one or more unnatural amino acids may be incorporated into atargeting agent in response to an amber codon. The one or more unnaturalamino acids may be site-specifically incorporated into a targetingagent.

Incorporating one or more unnatural amino acids into a targeting agentmay comprise use of one or more genetically encoded unnatural aminoacids with orthogonal chemical reactivity relative to the canonicaltwenty amino acids to site-specifically modify the targeting agent.Incorporating the one or more unnatural amino acids may comprise use ofan evolved tRNA/aminoacyl-tRNA synthetase pair to site-specificallyincorporate one or more unnatural amino acids at defined sites in thetargeting agent in response to one or more amber nonsense codon.

Additional methods for incorporating unnatural amino acids include, butare not limited to, methods disclosed in Chatterjee et al. (A VersatilePlatform for Single- and Multiple-Unnatural Amino Acid Mutagenesis inEscherichia coli, Biochemistry, 2013), Kazane et al. (J Am Chem Soc,135(1):340-6, 2013), Kim et al. (JAm Chem Soc, 134(24):9918-21, 2012),Johnson et al. (Nat Chem Biol, 7(11):779-86, 2011) and Hutchins et al.(J Mol Biol, 406(4):595-603, 2011).

VB. Coupling of Linkers

The methods disclosed herein may comprise coupling one or more linkersto one or more antibodies, antibody fragments, targeting agents, orcombinations thereof to produce one or more intermediates such as anantibody-linker intermediate, an antibody fragment-linker intermediateand/or a targeting agent antibody conjugate-linker intermediate. Themethods may comprise coupling a first linker to an antibody or antibodyfragment to produce an antibody-linker intermediate or antibodyfragment-linker intermediate. The methods may comprise coupling a linkerto a targeting agent to produce a targeting agent-linker intermediate.

Coupling of the one or more linkers to the antibody, antibody fragment,or targeting agent may occur simultaneously. Coupling of the one or morelinkers to the antibody, antibody fragment, or targeting molecule mayoccur sequentially. Coupling of the one or more linkers to the antibody,antibody fragment, or targeting molecule may occur in a single reactionvolume. Coupling of the one or more linkers to the antibody, antibodyfragment, or targeting molecule may occur in two or more reactionvolumes.

Coupling one or more linkers to the antibody, antibody fragment and/ortargeting molecule may comprise forming one or more oximes between thelinker and the antibody, antibody fragment or targeting molecule.Coupling one or more linkers to the antibody, antibody fragment and/ortargeting agent may comprise forming one or more stable bonds betweenlinker and the antibody, antibody fragment or targeting agent. Couplingone or more linkers to the antibody, antibody fragment and/or targetingagent may comprise forming one or more covalent bonds between linker andthe antibody, antibody fragment or targeting agent. Coupling one or morelinkers to the antibody, antibody fragment and/or targeting agent maycomprise forming one or more non-covalent bonds between linker and theantibody, antibody fragment or targeting agent. Coupling one or morelinkers to the antibody, antibody fragment and/or ligand may compriseforming one or more ionic bonds between linker and the antibody,antibody fragment or targeting agent.

Coupling one or more linkers to the antibody or antibody fragment maycomprise site specifically coupling one or more linkers to the antibodyor antibody fragment. Site-specific coupling may comprise linking theone or more linkers to the unnatural amino acid of the antibody orantibody fragment. Linking the one or more linkers to the unnaturalamino acid of the antibody or antibody fragment may comprise formationof an oxime. Linking the one or more linkers to the unnatural amino acidof the antibody or antibody fragment may comprise, by way ofnon-limiting example, reacting a hydroxylamine of the one or morelinkers with an aldehyde or ketone of a amino acid. The amino acid maybe an unnatural amino acid.

VC. Linking Antibodies, Antibody Fragments, and/or Targeting Agents

The methods may comprise linking the antibody, antibody fragment,targeting agent or intermediates thereof to produce a targeting agentantibody conjugate comprising (a) an antibody or antibody fragment; (b)one or more linkers; and (c) a targeting agent, wherein the one or morelinkers link the first antibody or antibody fragment to the targetingagent. The method may further comprise conjugating the one or morelinkers to a targeting agent to produce a targeting agent-linkerintermediate (Y-L1 or L1-Y) and coupling the targeting agent-linkerintermediate to the antibody or antibody fragment. The method mayfurther comprise conjugating the one or more linkers to the antibody orantibody fragment to produce an antibody-linker intermediate or antibodyfragment-linker intermediate (X-L1 or L1-X) and coupling theantibody-linker intermediate or antibody-fragment-linker intermediate tothe targeting agent. Coupling an intermediate to an antibody, antibodyfragment or targeting agent may comprise formation of an oxime. Couplingan intermediate to an antibody, antibody fragment or targeting agent maycomprise formation of the oxime in an acidic solution. Coupling anintermediate to an antibody, antibody fragment or targeting agent maycomprise formation of the oxime in a slightly acidic solution. Couplingan intermediate to an antibody, antibody fragment or targeting agent maycomprise formation of the oxime in a slightly neutral solution. Theantibody or antibody fragment may comprise an unnatural amino acid.Linking the antibody or antibody fragment to the targeting agent-linkerintermediate may comprise forming an oxime between the unnatural aminoacid and the targeting agent-linker intermediate. The targeting agentmay comprise an unnatural amino acid. Linking the targeting agent to theantibody-linker intermediate or antibody fragment-linker intermediatemay comprise forming an oxime between the unnatural amino acid and theantibody-linker intermediate or the antibody fragment-linkerintermediate. The method of producing a targeting agent antibodyconjugate may comprise (a) conjugating a first linker (L1) to theantibody or antibody fragment to produce an antibody-linker intermediateor antibody fragment-linker intermediate (X-L1 or L1-X); (b) conjugatinga second linker (L2) to the targeting agent to produce a targetingagent-linker intermediate (Y-L2 or L2-Y); and (c) linking the twointermediates together to produce the targeting agent antibodyconjugate, X-L1-L2-Y or Y-L2-L-X. Conjugating the linker to theantibody, antibody fragment or targeting agent may comprise productionof an ionic bond, a covalent bond, a non-covalent bond or a combinationthereof between the linker and the antibody, antibody fragment ortargeting agent. Conjugating the linker to the antibody, antibodyfragment or targeting agent may be performed as described in Roberts etal., Advanced Drug Delivery Reviews 54:459-476 (2002). L1 and/or L2 maycomprise a linker selected from a bifunctional linker, a cleavablelinker, a non-cleavable linker, an ethylene glycol linker, abifunctional ethylene glycol linker, a flexible linker, or an inflexiblelinker. L1 and/or L2 may comprise a linker selected from the groupcomprising cyclooctyne, cyclopropene, arly/alkyl azides,trans-cyclooctene, norborene, and tetrazines. A terminus of L1 and/or aterminus of L2 may comprise an alkoxy-amine. A terminus of L1 and/or aterminus of L2 may comprise an azide or cyclooctyne group. X may becoupled to L1 by a chemical group selected from a cyclooctyne,cyclopropene, arly/alkyl azide, trans-cyclooctene, norborene, andtetrazine. Linking the antibody-linker intermediate or antibodyfragment-linker intermediate (X-L¹ or L¹-X) and targeting agent-linkerintermediate (Y-L2 or L2-Y) may comprise conducting one or morecopper-free reactions. Linking the antibody-linker intermediate orantibody fragment-linker intermediate (X-L1 or L1-X) and targetingagent-linker intermediate (Y-L2 or L2-Y) may comprise conducting one ormore copper-containing reactions. Linking the antibody-linkerintermediate or antibody fragment-linker intermediate (X-L1 or L1-X) andtargeting agent-linker intermediate (Y-L2 or L2-Y) may comprise one ormore cycloadditions. Linking the antibody-linker intermediate orantibody fragment-linker intermediate (X-L1 or L1-X) and targetingagent-linker intermediate (Y-L2 or L2-Y) may comprise one or moreHuisgen-cycloadditions. Linking the antibody-linker intermediate orantibody fragment-linker intermediate (X-L1 or L1-X) and targetingagent-linker intermediate (Y-L2 or L2-Y) may comprise one or more DielsAlder reactions. Linking the antibody-linker intermediate or antibodyfragment-linker intermediate (X-L1 or L1-X) and targeting agent-linkerintermediate (Y-L2 or L2-Y) may comprise one or more Hetero Diels Alderreaction.

VD. Purification of Antibodies

The methods may further comprise purifying the targeting agent antibodyconjugate comprising (a) an antibody or antibody fragment comprising oneor more unnatural amino acids; (b) a targeting agent; and (c) one ormore linkers, wherein the one or more linkers link the antibody orantibody fragment to the targeting agent. The methods may furthercomprise purifying one or more intermediates of the antibody, antibodyfragment or targeting agent (e.g., antibody-linker, antibodyfragment-linker, or ligand-linker molecule). Purifying the antibody orintermediates may comprise removal of excess linkers, non-linkedantibodies, non-linked antibody fragments or non-linked ligands.Purifying the antibody or intermediates may comprise use of one or moreconcentrator columns, electrophoresis, filtration, centrifugation,chromatography or a combination thereof. Chromatography may comprisesize-exclusion chromatography. Additional chromatography methodsinclude, but are not limited to, hydrophobic interaction chromatography,ion exchange chromatography, affinity chromatography, metal binding,immunoaffinity chromatography, and high performance liquidchromatography or high pressure liquid chromatography. Electrophoresismay comprise denaturing electrophoresis or non-denaturingelectrophoresis.

Antibodies, antibody fragments, targeting agents or intermediates maycomprise one or more tags. The linkers may comprise one or more tags.The tags may be used to purify the antibodies, antibody fragments,targeting agents or intermediates. The one or more tags may be cleavedby one or more proteases. Examples of tags include, but are not limitedto, polyhistidine, FLAG, HA, c-myc, V5, chitin binding protein (CBP),maltose binding protein (MBP), and glutathione-S-transferase (GST).

The methods may further comprise lyophilization or ultracentrifugationof the antibodies, antibody fragments, targeting agents orintermediates.

The purity of the antibody or antibody fragment may be equal to orgreater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more. The purity of the antibody may beequal to or greater than 85%. The purity of the antibody or antibodyfragment may be equal to or greater than 90%. The purity of the antibodyor antibody fragment may be equal to or greater than 95%. The purity ofthe antibody or antibody fragment may be equal to or greater than 97%.

The purity of the intermediate (e.g., antibody-linker, antibodyfragment-linker, targeting agent-linker) may be equal to or greater than50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more. The purity of the intermediate may be equalto or greater than 85%. The purity of the intermediate may be equal toor greater than 90%. The purity of the intermediate may be equal to orgreater than 95%. The purity of the intermediate may be equal to orgreater than 97%.

The purity of the targeting agent may be equal to or greater than 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more. The purity of the targeting agent may be equal toor greater than 85%. The purity of the targeting agent may be equal toor greater than 90%. The purity of the targeting agent may be equal toor greater than 95%. The purity of the targeting agent may be equal toor greater than 97%.

The homogeneity of the antibody or antibody fragment may be equal to orgreater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more. The homogeneity of the antibody orantibody fragment may be equal to or greater than 85%. The homogeneityof the antibody or antibody fragment may be equal to or greater than90%. The homogeneity of the antibody or antibody fragment may be equalto or greater than 95%. The homogeneity of the antibody or antibodyfragment may be equal to or greater than 97%.

The homogeneity of the intermediate (e.g., antibody-linker, antibodyfragment-linker, targeting agent-linker) may be equal to or greater than50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more. The homogeneity of the antibody may be equalto or greater than 85%. The homogeneity of the antibody may be equal toor greater than 90%. The homogeneity of the antibody may be equal to orgreater than 95%. The homogeneity of the antibody may be equal to orgreater than 97%.

The homogeneity of the targeting agent may be equal to or greater than50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more. The homogeneity of the targeting agent maybe equal to or greater than 85%. The homogeneity of the targeting agentmay be equal to or greater than 90%. The homogeneity of the targetingagent may be equal to or greater than 95%. The homogeneity of thetargeting agent may be equal to or greater than 97%.

VI. Cells

The targeting agent antibody conjugates disclosed herein may bind to oneor more receptors, co-receptors, antigens, or cell markers on one ormore cells. The targeting agent antibody conjugates disclosed herein maycomprise (a) an antibody or antibody fragment and (b) a targeting agent,wherein (i) the antibody or antibody fragment binds to or interacts witha receptor, co-receptor, antigen or cell marker on a first cell; (ii)the targeting agent binds to or interacts with a receptor, co-receptor,antigen or cell marker on a second cell; or (iii) a combination of (i)and (ii), and wherein (iv) the antibody or antibody fragment comprisesone or more unnatural amino acids; (v) the targeting agent comprises oneor more unnatural amino acids; or (vi) a combination of (iv) and (v).The first cell may be a cytotoxic effector cell. The second cell may bea target cell. Generally, binding of the cytotoxic effector cell and thetarget cell to the targeting agent antibody construct brings the targetcell into a proximity with the cytotoxic effector cell that issufficiently close for an activity of the cytotoxic effector cell tohave an effect on the target cell. For example, when the cytotoxiceffector cell and the target cell are bound to the targeting agentantibody conjugate, the cytotoxic effector cell may release cytokinesthat bind to cytokine receptors on the target cell.

The cytotoxic effector cell may a cytotoxic cell. The cytotoxic cell maybe a hematopoietic cell. The hematopoietic cell may be a macrophage, aneutrophil, an eosinophil, a NK cell, a B-cell, or a T-cell. Thehematopoietic cell may be a T cell. The T cell may be a cytotoxic Tcell. The T cell may be a natural killer T cell.

The target cell may be a cancerous cell. The target cell may be a tumorcell. The target cell may be a leukemic cell. The target cell may be alymphomic cell. The target cell may be a metastatic cell. The targetcell may be genetically modified. The target cell may comprise a geneticmutation. The genetic mutation may comprise an oncogenic mutation. Thegenetic mutation may be a mutation of a tumor suppressor gene. Thegenetic mutation may be a mutation of a protooncogene. The target cellmay be an inflammatory cell. The target cell may be an infected cell.The target cell may be a pathogenic cell.

The first cell and the second cell may be the same type of cell. Thefirst cell and the second cell may be different cell types.Alternatively, the first cell and the second cell may be the same cell.The targeting agent antibody conjugate may bind to or interact with areceptor, co-receptor, antigen or cell marker on more than two differentcell types. The antibody or antibody fragment may bind to or interactwith a receptor, co-receptor, antigen or cell marker on two or moredifferent cell types. The targeting agent may bind to or interact with areceptor, co-receptor, antigen or cell marker on two or more differentcell types. The different cell types may differ by their cell lineage.The different cell types may differ by their function. The differentcell types may differ by the expression of one or more proteins. Thedifferent cell types may differ by their morphology. The different celltypes may differ by their locations. The different cell types may differby their genotype. The different cell types may differ by a singlegenetic mutation. The different cell types may differ by a more than onegenetic mutation.

The one or more cells may comprise hematopoietic cells. Hematopoieticcells include, but are not limited to, basophilic myelocytes, basophils,B-cells, burst forming unit erythroid (BFU-E), burst forming unitmegakaryocytes (BFU-Mk), colony forming unit basophils (CFU-Bas), colonyforming unit erythroid (CFU-E), colonly forming unit eosinophils(CFU-Eo), colony forming unit granulocytes (CFU-G), colony forming unitgranulocyte erythrocyte monocyte maccrophage (CFU-GEMM), colony formingunit granulocyte maccrophage (CFU-GM), colony forming unitemegakaryocyte (CFU-Mk, CFU-MEG), common dendritic progenitor, commonlymphoid progenitor cells, common myeloerythroid progenitors, commonmyeloid progenitors, common myelolymphoid progenitors, double negative 1(DN1) cells, DN2 cells, DN3 cells, DN4 cells, double-positive cells (DPcells), eosinophilic myelocytes, eosinophils, erythrocytes, lymphoidstem cells, lymphoid-related dendritic cells, macrophages, mast cells,megakaryocytes, memory B-cells, memory cells, memory T-cells,monoblasts, monocytes, myeloblasts, myeloid stem cells, myeloid-relateddendritic cells, neutrophilic myelocytes, neutrophils, natural killercells (NK-cells), natural killer T-cells (NKT-cells), platelets,pro-B1-cells, pro-B-2-cells, pro-B-cells, proerythroblasts,promonocytes, regulatory T-cells (Tregs), T-cells, T-helper (Th) cells,Th0 cells, Th1 cells, Th2 cells, Th3 cells, Th17 cells. BFU-E or CFU-Emay refer to erythroid precursor cells that may differentiate intoerythrocytes. CFU-E cells may be more developed than BFU-E cells. CFU-Eomay refer to developmental type of blood-forming cells that may developinto eosinophils. CFU-G may refer to a developmental type ofblood-forming cells that may be a precursor of granulocytes. CFU-GEMMmay refer to a pluripotent type of precursor cell in the lineage ofblood-forming cells that may differentiate into granuclocytes,erythrocytes, monocytes and/or macrophages. CFU-GM may refer to apluropotent type of precursor cell in the lineage of blood-forming cellsthat may differentiate into granuloctyes and/or macrophages. BFU-Mk,CFU-Mk or CFU-MEG may refer to precursor cells that may differentiateinto megakaryocytes. CFU-Mk or CFU-MEG cells may be more developed thanBFU-Mk cells.

The one or more cells may be from an organ or tissue. The organ may be agland organ. The organ may be an organ of the digestive or endocrinesystem. The organ may be both an endocrine gland and a digestive organ.The organ may be derived from endoderm, ectoderm, primitive endoderm, ormesoderm. The organ may be an adrenal gland. In some cases, the adrenalgland comprises chromaffin cells or ganglion cells. Alternatively, theorgan is an appendix, bladder, or brain. In some cases, the braincomprises neurons (e.g., nerve cells) or glial cells. Glial cellsinclude, but are not limited to, astrocytes, oligodendrocytes, andependymal cells. In some instances, the organ is an ear, esophagus, eye,or gallbladder. The gallbladder comprises cholecystocytes. The organ maybe a kidney. The kidney may comprise a kidney glomerulus parietal cell,kidney glomerululs podocyte, kidney proximal tubule brush border cell,Loop of Henle thin segment cell, thick ascending limb cell, kidneydistal tubule cell, kidney collecting ductal cell, or interstitialkidney cell. In some instances, the organ is a large intestine and thelarge intestine may comprise enterocytes, goblet cells, caveolated tuftcells, enteroendocrine cells, or ganglion neurons. The organ may be aliver. The liver may comprise parenchymal or non-parenchymal cells.Examples of parenchymal cells comprise hepatocytes. Non-parenchymalcells include, but are not limited to, sinusoidal endothelial cells,kupffer cells and hepatic stellate cells. In some instances, the organis a lung, mouth, nose, parathyroid gland, pineal gland, pituitarygland, skin, small intestine, stomach, spleen, thymus, thyroid gland,trachea, uterus, or vermiform appendix. In some instances, the organ maybe a heart. In some instances, the heart comprises cardiomyocytes. Insome instances, the organ is a muscle (e.g., heart muscle, skeletalmuscle, smooth muscle, etc.). The muscle may comprise myocytes.

In some cases, the cells are from a tissue. The tissue may be aconnective tissue, epithelial tissue, muscular tissue, or nervoustissue. Alternatively, the tissue is a bone, tendon (both referred to asmusculoskeletal grafts), cornea, skin, heart valve, or vein.

Connective tissue may be a fibrous tissue and is often found throughoutthe body. Examples of connective tissues include, but are not limitedto, connective tissue, fat tissue, dense fibrous tissue, cartilage,bone, blood, and lymph. Generally, connective tissue has three maincomponents: cells, fibers, and extracellular matrix, which may beembedded in the body fluids. Fibroblasts are often the cells responsiblefor the production of connective tissue. The interaction of the fibers,the extracellular matrix and the water, together, may form the pliableconnective tissue as a whole. Connective tissue may make up a variety ofphysical structures including tendons and the connective framework offibers in muscles, capsules and ligaments around joints, cartilage,bone, adipose tissue, blood and lymphatic tissue. Connective tissue (CT)may be classified into three subtypes: embryonic CT, proper CT, andspecial CT. The proper CT subtype may include dense regular CT, denseirregular CT, and loose CT. The special CT subtype may includecartilage, bone, adipose tissue, blood, hematopoietic tissue andlymphatic tissue.

Often connective tissues have distinct functions, characteristics, andcompositions. The functions of connective tissue may include storage ofenergy, protection of organs, providing structural framework for thebody, and connection of body tissues. The connective tissue may becharacterized by cells that are spread through an extracellular fluid.In some instances, the connective tissue may comprise a groundsubstance, which is often a clear, colorless, and viscous fluidcontaining glycosaminoglycans and proteoglycans. The ground substancemay fix the bodywater and the collagen fibers in the intercellularspaces. Ground substance may also slow the spread of pathogens.

The connective tissue may be fibrous and the fibrous tissue may comprisedistinct compositions and be localized to specific areas of the body.For example, collagenous fibers often contain alpha polypeptide chainsand may be primarily localized to a tendon, ligament, skin, cornea,cartilage, bone, blood vessels, gut, and intervertebral disc. In anotherexample, elastic fibers may comprise elastic microfibrill and elastinand may be primarily localized to an extracellular matrix. Reticularfibers are another example of fibrous tissue and may be localized to theliver, bone marrow, or lymphatic organs.

However, not all types of connective tissues are fibrous. Examples ofnon-fibrous connective tissues are adipose tissue and blood. Adiposetissue may provide a “mechanical cushioning” to our body. Although thereis often no dense collagen network in adipose tissue, groups of adiposecells may be kept together by collagen fibers and collagen sheets inorder to keep fat tissue under compression in place (for example thesole of the foot).

Epithelia are tissues that may consist of closely apposed cells withoutintervening intercellular substances. Epithelia are often avascular, butepithelia may “grow” on an underlying layer of vascular connectivetissue. The connective tissue and the epithelium may be separated by abasement membrane. Epithelium may cover all free surfaces of the body.Epithelium may also line the large internal body cavities, where it istermed mesothelium. Furthermore, the internal surfaces of blood andlymph vessels may be lined by epithelium, here called endothelium.Epithelia are often classified on the basis of the number of cell layersand the shape of the cells in the surface layer. If there is only onelayer of cells in the epithelium, it is designated simple. If there aretwo or more layers of cells, it is termed stratified. Cells in thesurface layer may be described according to their height as squamous(scale- or plate-like), cuboidal or columnar.

Different types of epithelial tissues may have specialized functions andlocations within the body. For example, pseudostratified columnar mayfunction to remove dust and particles from airways and may have cilia.The pseudostratified columnar may line the respiratory passageways. Thesimple columnar may be involved in absorption and often line the uterusand most organs of the digestive tract. The simple cuboidal may beinvolved in secretion and absorption and may be localized to glands,kidney tubules, and ovaries. The simple squamous may play a role indiffusion and filtration and may be localized to lungs, walls ofcapillaries and vessels. The stratified squamous may protect underlyingcells and is often localized to the skin, throat, vagina, and mouth. Thestratified cuboidal may be involved in protection and may line ducts ofthe mammary glands, sweat glands, and pancreas. The stratified columnarmay be involved in protection and secretion and may be localized to themale urethra and vas deferens, and parts of the pharynx.

Muscular tissue is often a contractile tissue and may be derived fromthe mesodermal layer of embryonic germ cells. Muscle cells may containcontractile filaments that move past each other and change the size ofthe cell. They are classified as skeletal, cardiac, or smooth muscles.Skeletal muscle or “voluntary muscle” may be anchored by tendons (or byaponeuroses at a few places) to bone and may be used to effect skeletalmovement such as locomotion and in maintaining posture. Smooth muscle or“involuntary muscle” is often found within the walls of organs andstructures such as the esophagus, stomach, intestines, bronchi, uterus,urethra, bladder, blood vessels, and the arrector pill in the skin (inwhich it controls erection of body hair). Cardiac muscle is also an“involuntary muscle” but may be more structurally similar to skeletalmuscle, and is often found in the heart.

Cardiac and skeletal muscles are often “striated” in that they containsarcomeres and are packed into highly regular arrangements of bundles.While skeletal muscles may be arranged in regular, parallel bundles,cardiac muscle often connects at branching, irregular angles (calledintercalated discs). Striated muscle may contract and relax in short,intense bursts, whereas smooth muscle may sustain longer or evennear-permanent contractions.

Skeletal muscle may be divided into several subtypes. Type I, slowoxidative, slow twitch, or “red” muscle is often dense with capillariesand may be rich in mitochondria and myoglobin, giving the muscle tissueits characteristic red color. It may carry more oxygen and sustainaerobic activity. Type II, fast twitch muscle, has three major kinds,Type IIa, Type IIx, and Type IIb. Type IIa is often aerobic and may berich in mitochondria and capillaries and may appear red. Type IIx (alsoknown as type IId), which is often less dense in mitochondria andmyoglobin. Type IIb, which may be anaerobic, glycolytic, “white” musclethat is often even less dense in mitochondria and myoglobin.

Nervous tissue is one of four major classes of tissue. Nervous tissue isoften the main component of the nervous system, the brain, spinal cord,and nerves, which may regulate and control body functions. Nervoustissue is often composed of neurons and the neuroglia cells. Neurons maytransmit impulses. Neuroglial cells may assist in propagation of thenerve impulse as well as provide nutrients to the neuron. Nervous tissueis often made of nerve cells that may come in many varieties, all ofwhich may be distinctly characterized by the axon or long stem like partof the cell that sends action potential signals to the next cell.

Functions of the nervous system may include sensory input, integration,controls of muscles and glands, homeostasis, and mental activity.Nervous tissue may react to stimuli and may conduct impulses to variousorgans in the body which often bring about a response to the stimulus.Nerve tissue (as in the brain, spinal cord and peripheral nerves thatbranch throughout the body) are often made up of specialized nerve cellscalled neurons. Neurons are easily stimulated and transmit impulses veryrapidly. A nerve often comprises many nerve cell fibers (neurons) boundtogether by connective tissue. A sheath of dense connective tissue, theepineurium may surround the nerve. This sheath penetrates the nerve toform the perineurium which surrounds bundles of nerve fibers. Bloodvessels of various sizes may be seen in the epineurium. The endoneurium,which consists of a thin layer of loose connective tissue, surrounds theindividual nerve fibers.

The cell body may be enclosed by a cell (plasma) membrane and may have acentral nucleus. Granules called Nissl bodies are often found in thecytoplasm of the cell body. Within the cell body, extremely fineneurofibrils may extend from the dendrites into the axon. The axon isoften surrounded by the myelin sheath, which forms a whitish,non-cellular, fatty layer around the axon. Outside the myelin sheath maybe a cellular layer called the neurilemma or sheath of Schwann cells.The myelin sheath together with the neurilemma is also known as themedullary sheath. This medullary sheath may be interrupted at intervalsby the nodes of Ranvier.

Neurons may be classified both structurally and functionally. Structuralclassification may group neurons according to the number of processesextending from their cell body. Three major neuron groups often make upthis classification: multipolar (polar=end, pole), bipolar and unipolarneurons. Multipolar neurons often have three or more processes. Theseare the most common neuron type in humans (more than 99% of neuronsbelong to this class) and the major neuron type in the CNS. Bipolarneurons are often spindle-shaped, with a dendrite at one end and an axonat the other. An example may be found in the light-sensitive retina ofthe eye. Unipolar neurons often comprise sensory neurons. Sensoryneurons normally have only a single process or fibre which divides closeto the cell body into two main branches (axon and dendrite).

The cells may also comprise hair follicles, hair cells, ear hair cells,ear hair stem cells, or cochlear cells. Hair cells are often the sensoryreceptors of both the auditory system and the vestibular system. Theauditory hair cells may be located within the organ of Corti on a thinbasilar membrane in the cochlea of the inner ear. Cochlear hair cellsmay come in two anatomically and functionally distinct types: the outerand inner hair cells.

The one or more cells may be a pathogenic cell. Pathogenic cellsinclude, but are not limited to, bacteria, viruses, fungi, andprotozoans. Examples of pathogens may include, but are not limited to,the bacteria, viruses, fungi, and protozoans disclosed herein.

VII. Receptors, Co-Receptors, Antigens and Cell Markers

The targeting agent antibody conjugates disclosed herein may bind to oneor more receptors, co-receptors, antigens, or cell markers on one ormore cells. The targeting agent antibody conjugates disclosed herein maycomprise (a) an antibody or antibody fragment and (b) targeting agent,wherein (i) the antibody or antibody fragment binds to or interacts witha receptor, co-receptor, antigen or cell marker on a first cell; (ii)the targeting agent binds to or interacts with a receptor, co-receptor,antigen or cell marker on a second cell; or (iii) a combination of (i)and (ii), and wherein (iv) the antibody or antibody fragment comprisesone or more unnatural amino acids; (v) the targeting agent comprises oneor more unnatural amino acids; or (vi) a combination of (iv) and (v).The first cell and the second cell may be the same type of cell. Thefirst cell and the second cell may be different cell types.Alternatively, the first cell and the second cell may be the same cell.

For example, for a targeting agent antibody conjugate of Formula I, IA,II, and/or IIA, X comprises an antibody or antibody fragment that maybinds to a receptor, co-receptor, antigen, trans-membrane protein orcell marker. Alternatively, or additionally, for a targeting agentantibody conjugate of Formula I, IA, II, and/or IIA, Y comprises atargeting agent that may bind to a receptor, co-receptor, antigen orcell marker.

Examples of receptors, co-receptors, antigens or cell markers mayinclude, but are not limited to, a receptor, co-receptor, antigen orcell marker on a hematopoietic cell, tissue cell, epithelial cell,mesothelial cell, dermal cell, endothelial cell, dendritic cell,vascular cell, stromal cell, neuron, cancer cell, bacteria, fungus, orvirus. The receptors, co-receptors, antigens or cell markers may beselected from the group comprising MUC16, GPNMB, Cripto, ED-8, TMEFF2,EphB2, EphA2, FAP, mesothelin, TAG-72, GD2, CAIX, and 5T4.

The receptor, co-receptor, antigen or cell marker may be overexpressedon a prostate cancer cell. The receptors, co-receptors, antigens or cellmarkers may comprise an endothelin. The receptor, co-receptor, antigenor cell marker may be PSMA.

The receptor may be a G-protein coupled receptor (GPCR). The receptormay be a tyrosine kinase receptor. The receptor may be a cytokinereceptor. The receptor may be a chemokine receptor. The receptor may bea growth factor receptor. The growth factor receptor may be an epidermalgrowth factor receptor (EGFR), platelet derived growth factor receptor(PDGFR) or fibroblast growth factor receptor (FGFR). The EGFR may beEGFR1. The EGFR may be Her2. The receptor may be a cholecystokinin Breceptor. The receptor may be a gonadotropin-releasing hormone receptor.The receptor may be a somatostatin receptor. The receptor may besomatostatin receptor 2. The receptor may be a gastrin-releasing peptidereceptor. The receptor may be a neurokinin receptor. The may beneurokinin 1 receptor, also known as tachykinin 1 receptor. The receptormay be a melanocortin receptor. The receptor may be melanocortin 1receptor. The receptor may be a neurotensin receptor. The receptor maybe a neuropeptide Y receptor. The receptor may be an integrin. Theintegrin may be an av integrin. The integrin may be avB3 integrin.

The receptor, co-receptor, antigen or cell surface marker may comprise acluster of differentiation protein. The cluster of differentiationprotein may be selected from CD19, CD20, CD22, CD25, CD30, CD40, CD56,CD64, CD70, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340,The differentiation protein may comprise CD38.

The trans-membrane protein may comprise a glycoprotein. The glycoproteinmay be C-type lectin-like molecule-1 (CLL-1).

The receptor, co-receptor, antigen, trans-membrane protein or cellmarker may be a cell adhesion molecule. The cell adhesion molecule maybind a cell. The cell adhesion molecule may bind extracellular matrix.The receptor, co-receptor, antigen or cell surface marker may comprise agap junction protein.

An antigen may evoke the production of one or more antibodies. Anantigen may refer to a molecule or molecular fragment that may be boundby a major histocompatiblity complex (MHC) and presented to a T-cellreceptor. The term “antigen” may also refer to an immunogen. Animmunogen may provoke an adaptive immune response if injected on its owninto a subject. An immunogen may induce an immune response by itself. Anantigen may also refer to a hapten. A hapten may be a targeting agent.Generally, a hapten may induce an immune response when attached to alarger carrier molecule, such as a protein. Antigens may be proteins orpolysaccharides. Antigens may comprise parts (e.g., coats, capsules,cell walls, flagella, fimbrae, and toxins) of bacteria, viruses, andother microorganisms. Lipids and nucleic acids may be antigenic whencombined with proteins and polysaccharides. Antigens may includesuperantigens, T-dependent antigens and T-independent antigens.

Antigens may be exogenous antigens or endogeneous antigens. Exogeneousantigens are typically antigens that have entered the body from theoutside, for example by inhalation, ingestion, or injection. Someantigens may start out as exogenous antigens, and later becomeendogenous (for example, intracellular viruses). Intracellular antigensmay be released back into circulation upon the destruction of theinfected cell, again. Endogenous antigens may be antigens that have beengenerated within previously-normal cells as a result of normal cellmetabolism, or because of viral or intracellular bacterial infection.

Antigens may also include autoantigens. An autoantigen may be a normalprotein or complex of proteins (and sometimes DNA or RNA) that isrecognized by the immune system of patients suffering from a specificautoimmune disease. These antigens should, under normal conditions, notbe the target of the immune system, but, due to mainly genetic andenvironmental factors, the normal immunological tolerance for such anantigen has been lost in these patients.

Antigens may include tumor antigens. Tumor antigens or neoantigens maybe antigens that are presented by MHC I or MHC II molecules on thesurface of tumor cells. These antigens may sometimes be presented bytumor cells and never by the normal ones. In this case, they are calledtumor-specific antigens (TSAs) and, in general, result from atumor-specific mutation. More common are antigens that are presented bytumor cells and normal cells, and they are called tumor-associatedantigens (TAAs). Cytotoxic T lymphocytes that recognize these antigensmay be able to destroy the tumor cells before they proliferate ormetastasize. Tumor antigens may also be on the surface of the tumor inthe form of, for example, a mutated receptor, in which case they may berecognized by B cells.

VIII. Indications

The targeting agent antibody conjugates and compositions disclosedherein may be used to treat one or more diseases or conditions in asubject in need thereof. The method may comprise administering atargeting agent antibody conjugate to a subject in need thereof. Thetargeting agent antibody conjugate may comprise (a) a first regioncomprising an antibody or antibody fragment that interacts with asurface marker on a first cell; and (b) a second region comprising anon-antibody region that interacts with a surface marker on a secondcell. The antibody or antibody fragment may comprise an anti-CD3antibody. The non-antibody region may interact with a PSMA on the secondcell. The non-antibody region may comprise DUPA. The targeting agentantibody conjugate may further comprise one or more linkers. The one ormore linkers may connect the first region and the second region. The oneor more diseases or conditions may be a cancer, a pathogenic infection,autoimmune disease, inflammatory disease, or genetic disorder.

In some instances, the one or more diseases comprises a cancer. Thecancer may comprise a recurrent and/or refractory cancer. Examples ofcancers include, but are not limited to, sarcomas, carcinomas, lymphomasor leukemias.

The cancer may comprise a neuroendocrine cancer. The cancer may comprisea pancreatic cancer. The cancer may comprise an exocrine pancreaticcancer. The cancer may comprise a thyroid cancer. The thyroid cancer maycomprise a medullary thyroid cancer.

The cancer may comprise a prostate cancer. The prostate cancer may be aPSMA-positive prostate cancer. PSMA expression may be highly upregulatedand restricted to cancer cells in some or all stages of the prostatecancer. The cancer may be hormone-refractory prostate cancer.

The cancer may comprise an epithelial cancer. The cancer may comprise abreast cancer. The cancer may comprise an endometrial cancer. The cancermay comprise an ovarian cancer. The ovarian cancer may comprise astromal ovarian cancer. The cancer may comprise a cervical cancer.

The cancer may comprise a skin cancer. The skin cancer may comprise aneo-angiogenic skin cancer. The skin cancer may comprise a melanoma.

The cancer may comprise a kidney cancer.

The cancer may comprise a lung cancer. The lung cancer may comprise asmall cell lung cancer. The lung cancer may comprise a non-small celllung cancer.

The cancer may comprise a colorectal cancer. The cancer may comprise agastric cancer. The cancer may comprise a colon cancer.

The cancer may comprise a brain cancer. The brain cancer may comprise abrain tumor. The cancer may comprise a glioblastoma. The cancer maycomprise an astrocytoma.

The cancer may comprise a blood cancer. The blood cancer may comprise aleukemia. The leukemia may comprise a myeloid leukemia. The cancer maycomprise a lymphoma. The lymphoma may comprise a non-Hodgkin's lymphoma.

The cancer may comprise a sarcoma. The sarcoma may comprise an Ewing'ssarcoma.

Sarcomas are cancers of the bone, cartilage, fat, muscle, blood vessels,or other connective or supportive tissue. Sarcomas include, but are notlimited to, bone cancer, fibrosarcoma, chondrosarcoma, Ewing's sarcoma,malignant hemangioendothelioma, malignant schwannoma, bilateralvestibular schwannoma, osteosarcoma, soft tissue sarcomas (e.g. alveolarsoft part sarcoma, angiosarcoma, cystosarcoma phylloides,dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, and synovial sarcoma).

Carcinomas are cancers that begin in the epithelial cells, which arecells that cover the surface of the body, produce hormones, and make upglands. By way of non-limiting example, carcinomas include breastcancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer,rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostatecancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer,vulvar cancer, uterine cancer, oral cancer, penile cancer, testicularcancer, esophageal cancer, skin cancer, cancer of the fallopian tubes,head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma,cutaneous or intraocular melanoma, cancer of the anal region, cancer ofthe small intestine, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, cancer of the urethra, cancer of the renal pelvis, cancer of theureter, cancer of the endometrium, cancer of the cervix, cancer of thepituitary gland, neoplasms of the central nervous system (CNS), primaryCNS lymphoma, brain stem glioma, and spinal axis tumors. In someinstances, the cancer is a skin cancer, such as a basal cell carcinoma,squamous, melanoma, nonmelanoma, or actinic (solar) keratosis.

In some instances, the cancer is a lung cancer. Lung cancer may start inthe airways that branch off the trachea to supply the lungs (bronchi) orthe small air sacs of the lung (the alveoli). Lung cancers includenon-small cell lung carcinoma (NSCLC), small cell lung carcinoma, andmesotheliomia. Examples of NSCLC include squamous cell carcinoma,adenocarcinoma, and large cell carcinoma. The mesothelioma may be acancerous tumor of the lining of the lung and chest cavitity (pleura) orlining of the abdomen (peritoneum). The mesothelioma may be due toasbestos exposure. The cancer may be a brain cancer, such as aglioblastoma.

Alternatively, the cancer may be a central nervous system (CNS) tumor.CNS tumors may be classified as gliomas or nongliomas. The glioma may bemalignant glioma, high grade glioma, diffuse intrinsic pontine glioma.Examples of gliomas include astrocytomas, oligodendrogliomas (ormixtures of oligodendroglioma and astocytoma elements), and ependymomas.Astrocytomas include, but are not limited to, low-grade astrocytomas,anaplastic astrocytomas, glioblastoma multiforme, pilocytic astrocytoma,pleomorphic xanthoastrocytoma, and subependymal giant cell astrocytoma.Oligodendrogliomas include low-grade oligodendrogliomas (oroligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomasinclude meningiomas, pituitary adenomas, primary CNS lymphomas, andmedulloblastomas. In some instances, the cancer is a meningioma.

The leukemia may be an acute lymphocytic leukemia, acute myelocyticleukemia, chronic lymphocytic leukemia, or chronic myelocytic leukemia.Additional types of leukemias include hairy cell leukemia, chronicmyelomonocytic leukemia, and juvenile myelomonocytic leukemia.

Lymphomas are cancers of the lymphocytes and may develop from either Bor T lymphocytes. The two major types of lymphoma are Hodgkin'slymphoma, previously known as Hodgkin's disease, and non-Hodgkin'slymphoma. Hodgkin's lymphoma is marked by the presence of theReed-Sternberg cell. Non-Hodgkin's lymphomas are all lymphomas which arenot Hodgkin's lymphoma. Non-Hodgkin lymphomas may be indolent lymphomasand aggressive lymphomas. Non-Hodgkin's lymphomas include, but are notlimited to, diffuse large B cell lymphoma, follicular lymphoma,mucosa-associated lymphatic tissue lymphoma (MALT), small celllymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma,mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodalmarginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma(SMZL), extranodal marginal zone B cell lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, and lymphomatoidgranulomatosis.

The one or more diseases or conditions may be a pathogenic infection.Pathogenic infections may be caused by one or more pathogens. In someinstances, the pathogen is a bacterium, fungi, virus, or protozoan.

Exemplary pathogens include but are not limited to: Bordetella,Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila,Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella,Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio,or Yersinia. In some cases, the disease or condition caused by thepathogen is tuberculosis and the heterogeneous sample comprises foreignmolecules derived from the bacterium Mycobacterium tuberculosis andmolecules derived from the subject. In some instances, the disease orcondition is caused by a bacterium is tuberculosis, pneumonia, which maybe caused by bacteria such as Streptococcus and Pseudomonas, a foodborneillness, which may be caused by bacteria such as Shigella, Campylobacterand Salmonella, and an infection such as tetanus, typhoid fever,diphtheria, syphilis and leprosy. The disease or condition may bebacterial vaginosis, a disease of the vagina caused by an imbalance ofnaturally occurring bacterial flora. Alternatively, the disease orcondition is a bacterial meningitis, a bacterial inflammation of themeninges (e.g., the protective membranes covering the brain and spinalcord). Other diseases or conditions caused by bacteria include, but arenot limited to, bacterial pneumonia, a urinary tract infection,bacterial gastroenteritis, and bacterial skin infection. Examples ofbacterial skin infections include, but are not limited to, impetigowhich may be caused by Staphylococcus aureus or Streptococcus pyogenes;erysipelas which may be caused by a streptococcus bacterial infection ofthe deep epidermis with lymphatic spread; and cellulitis which may becaused by normal skin flora or by exogenous bacteria.

The pathogen may be a fungus, such as, Candida, Aspergillus,Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Examples ofdiseases or conditions caused by a fungus include, but are not limitedto, jock itch, yeast infection, ringworm, and athlete's foot.

The pathogen may be a virus. Examples of viruses include, but are notlimited to, adenovirus, coxsackievirus, Epstein-Barr virus, Hepatitisvirus (e.g., Hepatitis A, B, and C), herpes simplex virus (type 1 and2), cytomegalovirus, herpes virus, HIV, influenza virus, measles virus,mumps virus, papillomavirus, parainfluenza virus, poliovirus,respiratory syncytial virus, rubella virus, and varicella-zoster virus.Examples of diseases or conditions caused by viruses include, but arenot limited to, cold, flu, hepatitis, AIDS, chicken pox, rubella, mumps,measles, warts, and poliomyelitis.

The pathogen may be a protozoan, such as Acanthamoeba (e.g., A.astronyxis, A. castellanii, A. culbertsoni, A. hatchetti, A. polyphaga,A. rhysodes, A. healyi, A. divionensis), Brachiola (e.g., B. connori, B.vesicularum), Cryptosporidium (e.g., C. parvum), Cyclospora (e.g., C.cayetanensis), Encephalitozoon (e.g., E. cuniculi, E. hellem, E.intestinalis), Entamoeba (e.g., E. histolytica), Enterocytozoon (e.g.,E. bieneusi), Giardia (e.g., G. lamblia), Isospora (e.g, I. belli),Microsporidium (e.g., M. africanum, M. ceylonensis), Naegleria (e.g., N.fowleri), Nosema (e.g., N. algerae, N. ocularum), Pleistophora,Trachipleistophora (e.g., T. anthropophthera, T. hominis), andVittaforma (e.g., V. corneae).

The disease or condition may be an autoimmune disease or autoimmunerelated disease. An autoimmune disorder may be a malfunction of thebody's immune system that causes the body to attack its own tissues.Examples of autoimmune diseases and autoimmune related diseases include,but are not limited to, Addison's disease, alopecia areata, ankylosingspondylitis, antiphospholipid syndrome (APS), autoimmune aplasticanemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmunemyocarditis, Behcet's disease, celiac sprue, Crohn's disease,dermatomyositis, eosinophilic fasciitis, erythema nodosum, giant cellarteritis (temporal arteritis), Goodpasture's syndrome, Graves' disease,Hashimoto's disease, idiopathic thrombocytopenic purpura (ITP), IgAnephropathy, juvenile arthritis, diabetes, juvenile diabetes, Kawasakisyndrome, Lambert-Eaton syndrome, lupus (SLE), mixed connective tissuedisease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus,polyarteritis nodosa, type I, II, & III autoimmune polyglandularsyndromes, polymyalgia rheumatica, polymyositis, psoriasis, psoriaticarthritis, Reiter's syndrome, relapsing polychondritis, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, sperm &testicular autoimmunity, stiff person syndrome, Takayasu's arteritis,temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis,vasculitis, vitiligo, and Wegener's granulomatosis.

The disease or condition may be an inflammatory disease. Examples ofinflammatory diseases include, but are not limited to, alveolitis,amyloidosis, angiitis, ankylosing spondylitis, avascular necrosis,Basedow's disease, Bell's palsy, bursitis, carpal tunnel syndrome,celiac disease, cholangitis, chondromalacia patella, chronic activehepatitis, chronic fatigue syndrome, Cogan's syndrome, congenital hipdysplasia, costochondritis, Crohn's Disease, cystic fibrosis, DeQuervain's tendinitis, diabetes associated arthritis, diffuse idiopathicskeletal hyperostosis, discoid lupus, Ehlers-Danlos syndrome, familialmediterranean fever, fascitis, fibrositis/fibromyalgia, frozen shoulder,ganglion cysts, giant cell arteritis, gout, Graves' Disease,HIV-associated rheumatic disease syndromes, hyperparathyroid associatedarthritis, infectious arthritis, inflammatory bowel syndrome/irritablebowel syndrome, juvenile rheumatoid arthritis, lyme disease, Marfan'sSyndrome, Mikulicz's Disease, mixed connective tissue disease, multiplesclerosis, myofascial pain syndrome, osteoarthritis, osteomalacia,osteoporosis and corticosteroid-induced osteoporosis, Paget's Disease,palindromic rheumatism, Parkinson's Disease, Plummer's Disease,polymyalgia rheumatica, polymyositis, pseudogout, psoriatic arthritis,Raynaud's Phenomenon/Syndrome, Reiter's Syndrome, rheumatic fever,rheumatoid arthritis, sarcoidosis, sciatica (lumbar radiculopathy),scleroderma, scurvy, sickle cell arthritis, Sjogren's Syndrome, spinalstenosis, spondyloisthesis, Still's Disease, systemic lupuserythematosis, Takayasu's (Pulseless) Disease, Tendinitis, tenniselbow/golf elbow, thyroid associated arthritis, trigger finger,ulcerative colitis, Wegener's Granulomatosis, and Whipple's Disease.

Further disclosed herein are uses of the targeting agent antibodyconjugate disclosed herein to treat a disease or condition. Disclosedherein is the use of a targeting agent antibody conjugate to treat acancer in a subject in need thereof, wherein the targeting agentantibody conjugate comprises (a) a first region comprising an antibodyor antibody fragment that interacts with an immune cell; and (b) asecond region comprising a non-antibody portion that interacts with atarget cell. The targeting agent antibody conjugate may further compriseone or more linkers. The one or more linkers may connect the firstregion and the second region of the targeting agent antibody conjugate.The non-antibody portion may interact with PSMA on the target cell. Thenon-antibody portion may comprise DUPA. The antibody or antibodyfragment may comprise an anti-CD3 antibody. The cancer may be a prostatecancer.

IX. Immune Modulation

The targeting agent antibody conjugates disclosed herein may be used tomodulate an immune response. Modulation of an immune response maycomprise stimulating, activating, increasing, enhancing, orup-regulating an immune response. Modulation of an immune response maycomprise suppressing, inhibiting, preventing, reducing, ordownregulating an immune response. For example, the antibodies maycomprise an antibody or antibody fragment that may bind to a first celland a targeting agent that may bind to a second cell. Binding of theantibody to the first and second cell may result in modulation of animmune response. The first cell may be an immune cell. The immune cellmay be a hematopoietic cell. The second cell may be an immune cell,healthy cell, cancer cell, bacteria, or virally-infected.

Methods of modulating an immune response may comprise (a) contacting animmune cell with a targeting agent antibody conjugate to produce animmune cell-targeting agent antibody conjugate complex; and (b)contacting a target cell with the immune cell-targeting agent antibodyconjugate complex, thereby modulating an immune response from the immunecell.

Alternatively, the method may comprise (a) contacting a target cell witha targeting agent antibody conjugate to produce a target cell-targetingagent antibody conjugate complex; and (b) contacting an immune cell withthe target cell-targeting agent antibody conjugate complex, therebymodulating an immune response from the immune cell. The targeting agentantibody conjugate may comprise an antibody or fragment thereof thatinteracts with a cell surface marker on the immune cell. The cellsurface marker on the immune may be a receptor or co-receptor. The cellsurface marker may be a CD3 T cell co-receptor. The cell surface markeron the immune cell may be a protein, glycoprotein, or steroid. Theimmune cell may be a T cell. The targeting agent antibody conjugate maycomprise a non-antibody portion that interacts with a cell surfacemarker on a target cell. The targeting agent antibody conjugate mayconnect the immune cell to the target cell. The target cell may be acancerous cell. The target cell may be a virally infected cell. Thetarget cell may be an immune cell. The immune cell may be a T cell. Theimmune cell may be a macrophage. The immune cell may be a B cell. Thecell surface marker on the target cell may be a receptor, co-receptor,surface protein, glycoprotein, or steroid. The non-antibody portion ofthe targeting antibody conjugate may comprise a ligand, cytokine, toxin,or small molecule. Methods of modulating the immune response may furthercomprise administering one or more additional targeting agent antibodyconjugates to the subject. The one or more additional targeting agentantibody conjugates may interact with one or more additional immunecells. The one or more additional immune cells may be the same type ofimmune cell as the previous immune cell. For example, a first targetingagent antibody conjugate may interact at with a T cell and a secondtargeting agent antibody conjugate may also interact with a T cell.Alternatively, or additionally, the one or more additional immune cellsmay be a different type of immune cell as the previous immune cell. Forexample, a first targeting agent antibody conjugate may interact with anantigen presenting cell and a second targeting agent antibody conjugatemay interact with a T cell. The one or more additional targeting agentantibody conjugates may interact with one or more additional cells. Theone or more additional cells may be of the same cell type as the immunecell or the target cell. For example, a first targeting agent antibodyconjugate may interact with a virally infected cell and a secondtargeting agent antibody conjugate may interact with a virally infectedcell. Alternatively, or additionally, the one or more additional cellsmay be different from the immune cell or target cell. For example, afirst targeting agent antibody conjugate may interact with a bacterialcell and a second targeting agent antibody conjugate may interact with avirally infected cell.

In another alternative, the method may comprise (a) contacting a samplecomprising a target cell and an immune cell with targeting agentantibody conjugate; and (b) connecting the target cell and the immunecell via the targeting agent antibody conjugate, thereby modulating animmune response from the immune cell. The targeting agent antibodyconjugate may comprise an antibody or fragment thereof that interactswith a cell surface marker on the immune cell. The cell surface markeron the immune may be a receptor or co-receptor. The cell surface markermay be a CD3 T cell co-receptor. The cell surface marker on the immunecell may be a protein, glycoprotein, or steroid. The immune cell may bea T cell. The targeting agent antibody conjugate may comprise anon-antibody portion that interacts with a cell surface marker on atarget cell. The targeting agent antibody conjugate may connect theimmune cell to the target cell. The target cell may be a cancerous cell.The target cell may be a virally infected cell. The target cell may bean immune cell. The immune cell may be a T cell. The immune cell may bea macrophage. The immune cell may be a B cell. The cell surface markeron the target cell may be a receptor, co-receptor, surface protein,glycoprotein, or steroid. The non-antibody portion of the targetingantibody conjugate may comprise a ligand, cytokine, toxin, or smallmolecule. Methods of modulating the immune response may further compriseadministering one or more additional targeting agent antibody conjugatesto the subject. The one or more additional targeting agent antibodyconjugates may interact with one or more additional immune cells. Theone or more additional immune cells may be the same type of immune cellas the previous immune cell. For example, a first targeting agentantibody conjugate may interact at with a T cell and a second targetingagent antibody conjugate may also interact with a T cell. Alternatively,or additionally, the one or more additional immune cells may be adifferent type of immune cell as the previous immune cell. For example,a first targeting agent antibody conjugate may interact with an antigenpresenting cell and a second targeting agent antibody conjugate mayinteract with a T cell. The one or more additional targeting agentantibody conjugates may interact with one or more additional cells. Theone or more additional cells may be of the same cell type as the immunecell or the target cell. For example, a first targeting agent antibodyconjugate may interact with a virally infected cell and a secondtargeting agent antibody conjugate may interact with a virally infectedcell. Alternatively, or additionally, the one or more additional cellsmay be different from the immune cell or target cell. For example, afirst targeting agent antibody conjugate may interact with a bacterialcell and a second targeting agent antibody conjugate may interact with avirally infected cell.

Methods of modulating an immune response may comprise administering acomposition comprising a targeting agent antibody conjugate to a subjectin need thereof, wherein the targeting agent antibody conjugatecomprises an antibody or antibody fragment that interacts with a cellsurface marker on an immune cell. The cell surface marker on the immunemay be a receptor or co-receptor. The cell surface marker may be a CD3 Tcell co-receptor. The cell surface marker on the immune cell may be aprotein, glycoprotein, or steroid. The immune cell may be a T cell. Thetargeting agent antibody conjugate may comprise a non-antibody portionthat interacts with a cell surface marker on a second cell. Thetargeting agent antibody conjugate may connect the immune cell to thesecond cell. The second cell may be a cancerous cell. The second cellmay be a virally infected cell. The second cell may be an immune cell.The immune cell may be a T cell. The immune cell may be a macrophage.The immune cell may be a B cell. The cell surface marker on the secondcell may be a receptor, co-receptor, surface protein, glycoprotein, orsteroid. The non-antibody portion of the targeting antibody conjugatemay comprise a ligand, cytokine, toxin, or small molecule. Methods ofmodulating the immune response may further comprise administering one ormore additional targeting agent antibody conjugates to the subject. Theone or more additional targeting agent antibody conjugates may interactwith one or more additional immune cells. The one or more additionalimmune cells may be the same type of immune cell as the previous immunecell. For example, a first targeting agent antibody conjugate mayinteract at with a T cell and a second targeting agent antibodyconjugate may also interact with a T cell. Alternatively, oradditionally, the one or more additional immune cells may be a differenttype of immune cell as the previous immune cell. For example, a firsttargeting agent antibody conjugate may interact with an antigenpresenting cell and a second targeting agent antibody conjugate mayinteract with a T cell. The one or more additional targeting agentantibody conjugates may interact with one or more additional cells. Theone or more additional cells may be of the same cell type as the immunecell or the second cell. For example, a first targeting agent antibodyconjugate may interact with a virally infected cell and a secondtargeting agent antibody conjugate may interact with a virally infectedcell. Alternatively, or additionally, the one or more additional cellsmay be different from the immune cell or second cell. For example, afirst targeting agent antibody conjugate may interact with a bacterialcell and a second targeting agent antibody conjugate may interact with avirally infected cell.

X. Additional Applications

Further disclosed herein are methods of connecting two or more cells.The method may comprise contacting a sample comprising two or more cellswith a targeting agent antibody conjugate. The targeting agent antibodyconjugate may comprise (a) a first region comprising an antibody orantibody fragment; and (b) a second region comprising a non-antibodyportion. The antibody or antibody fragment may interact with a surfacemarker on a first cell. The non-antibody portion may interact with asurface marker on a second cell. The first and second cells may be thesame type of cell. The first and second cells may be different types ofcells. The surface markers may be a receptor, co-receptor, protein,glycoprotein, antigen, polysaccharide, or steroid. The method mayfurther comprise contacting the sample with one or more additionaltargeting agent antibody conjugates. The one or more additionaltargeting agent antibody conjugates may comprise (a) a first regioncomprising an antibody or antibody fragment; and (b) a second regioncomprising a non-antibody portion. The antibody or antibody fragment ofthe one or more additional targeting agent antibody conjugates mayinteract with a surface marker on the first cell, the second cell, or athird cell. The non-antibody portion of the one or more additionaltargeting agent antibody conjugates may interact with a surface markeron the first cell, the second cell, or a fourth cell. The first, second,third, and/or fourth cells may be the same cell type. The first, second,third, and/or fourth cells may be different cell types.

The targeting agent antibody conjugates may be used to connect multiplecells. As such, multi-cell complexes may be formed by the attachment ofthe targeting agent antibody conjugates to the cells. Cell scaffoldsand/or cell matrices may be formed by the attachment of the targetingagent antibody conjugates to the cells.

The first region and second region of the targeting agent antibodyconjugates may be connected by one or more linkers. The linkers may bebiodegradable. The linkers may be cleavable. As such, temporarycell-cell complexes, scaffolds or matrices may be formed by usingtargeting agent antibody conjugates comprising biodegradable, cleavableor otherwise degradable linkers to connect two or more cells.Alternatively, or additionally, the linkers may be resistant to cleavageor degradation. As such, permanent or semi-permanent cell-cellcomplexes, scaffolds or matrices may be formed by using targeting agentantibody conjugates comprising cleavage or degradation-resistant linkersto connect two or more cells. The targeting agent antibody conjugatesmay comprise a mixture of degradable and non-degradable linkers tocreate flexible cell-cell complexes.

The use of the targeting agent antibody conjugates may enable analysisof cell-cell interactions and signal transduction pathways. The cellsmay further be isolated and expression profiling of the cells may beperformed. These type of analyses may be used in the diagnosis,prognosis, and/or treatment of a disease or condition in a subject inneed thereof.

EXAMPLES Example 1: Flow Cytometry Analysis: DUPA-Phthal-Double-αCD3Binding to C4-2 Cells

In this example, we conjugated two DUPA ligands to an anti-CD3 Fab suchthat the bivalent ligand may simultaneously bind each subunit of thePSMA homodimer with high avidity. To synthesize a bivalent Fab, weintroduced TAG codons at two different positions (light chain S202 andheavy chain K138), and the double mutant was expressed and purified asdescribed above. Notably, the expression levels were comparable to othersingle mutants and the wild type antibody that were previouslyexpressed. The double mutant antibody was conjugated with P-Phthal (3)or P-DNP (4) as described above; LC-MS analysis revealed that thereaction was complete (>95% efficiency) within 48 hours, yielding thebivalent conjugates, P-Phthal-double-αCD3 (12) and P-DNP-double-αCD3(13). After purification, the structures were confirmed by SDS-PAGE andLC-MS (the reaction yields after purifications are >90%). Binding of thebivalent conjugates to C4-2 cells was then assessed (together withmonovalent conjugates) using flow cytometry (FIG. 9, Table 1). Asignificant improvement was observed in binding affinity (>60-fold) forthe bivalent P-Phthal-double-αCD3 (12) compared to the monovalentP-Phthal-HK138-αCD3 (7). Interestingly, P-DNP-double-αCD3 (13) did notshow significant improvement over its monovalent equivalentP-DNP-HK138-αCD3 (8), which already had high affinity. The enhancedbinding affinity of P-Phthal-double-αCD3 (12) was particularlyencouraging because despite the high affinity of the DNP group, itsknown high immunogenicity might limit its use in vivo.

TABLE 1 FACS analysis of the binding of different P-anti-CD3 conjugatesto PSMA-positive C4-2 cells at various concentrations. Percent PositiveP-Phthal- P-Phthal- P-Phthal- P-Phthal- P-DNP- P-Pthal- P-DNP- [M]LC-109 LC-202 HC-123 HC-138 HC double double 0.000001 100 99.9 93.2 9999.9 99.8 99.6   2E−07 99.4 99.8 92.8 97.5 100 98 98.7   4E−08 95.2 95.669.7 89.5 99.5 96.2 97.7   8E−09 56.7 42.5 7.6 32.4 99 95.1 97.5 1.6E−091.94 1.37 0.42 0.82 93.7 91 96.9 3.2E−10 0.49 0.47 0.76 0.079 38.2 6086.3 6.4E−11 0.88 0.38 0.29 0.08 1.44 5.46 27.1 1.28E−11  0 0.66 0.520.43 0.13 0.4 0.81

Example 2: In Vitro Cytotoxicity of DUPA/Anti-CD3 Conjugates

We next compared the in vitro cytotoxicity of various DUPA/αCD3conjugates. Freshly purified human peripheral blood mononuclear cells(hPBMCs) were mixed with C4-2 (PSMA+) cells at a 10:1 ratio (100,000 and10,000 cells, respectively), and incubated with each conjugate for 24hours. A 1:1 mixture of wild-type UCHT1 Fab and the DUPA-linkerconjugate (P-DNP, 4) was used as a negative control. Cytotoxicity wasdetermined by measuring lactose dehydrogenase (LDH) released from lysedtarget cells. As shown in FIG. 10 and Table 2, each of the conjugates,with the exception of P-Phthal-HC-123-αCD3 (11) which poorly bound C4-2cells in the previous assay, showed dose dependent cytotoxicity.P-Phthal-LC-109-αCD3 (9) showed reduced cytotoxicity (EC₅₀˜4.1 nM)compared to P-Phthal-LC-202-αCD3 (10, EC₅₀˜0.4 nM) andP-Phthal-HC-138-αCD3 (7, EC₅₀ 0.5 nM), although all had similaraffinities in the binding studies. Finally, P-Phthal-doublea-CD3 (12)showed the highest cytotoxicity (ECso 0.1 nM) compared to the monovalentconstructs. No cytotoxicity was observed even at the highestconcentration measured with the unconjugated DUPA-linker and UCHT1 Fabmixures. Taken together, these results indicate that not only theaffinity but also the geometry of the bispecific significantly affectscytotoxicity. Based on the binding and in vitro cytotoxicity assays, wechose the P-Phthal-double-αCD3 (12) (also referred to as PSMA-targetingSmall Molecule Antibody Conjugate, P-targeting agent antibody conjugate)conjugate for further characterization.

TABLE 2 In vitro cytotoxicity of target cells Percent Cytotoxicity [pM]T109 S202 A123 0 −6.31405 −1.15703 8.561983 −3.33884 0.628099 1.619835−1.91715 4.861349 3.218076 1.28 −5.52066 2.016529 3.801653 −2.347112.809917 −1.95041 −2.12256 3.834303 4.450531 6.4 −6.5124 7.5702487.966942 −2.94215 3.206612 4.198347 −0.27388 0.342349 2.191031 32−4.92562 6.578512 6.380165 −2.94215 4.198347 3.404959 0.342349 3.0126676.710031 160 2.214876 9.752066 11.73554 10.54545 25.61983 17.8843−3.97124 −5.40911 5.272167 800 16.29752 35.33884 30.97521 52 63.7024856.95868 −3.97124 −0.06847 6.710031 4000 35.73554 47.83471 48.628164.29752 75.80165 76.3967 5.272167 4.450531 11.84526 20000 51.2066165.09091 75.20661 72.42975 83.1405 77.58678 8.764122 5.477576 15.54262100000 66.47934 76.19835 83.33884 83.1405 83.73553 82.54546 9.9965765.272167 16.15885 Percent Cytotoxicity [pM] K138 phthal double unconj 0−3.14961 −2.9442 −0.06847 −5.89878 7.504363 3.73473 0 0 0 1.28 −1.71174−0.06847 1.163985 2.268761 5.410122 3.73473 0 0.230681 0.230947 6.4−2.53338 1.369394 12.05067 −1.08202 3.525306 4.991274 −0.92593 −0.46136−0.57737 32 −2.12256 1.780212 4.245121 6.876091 11.90227 10.85515−0.61728 −0.69204 −1.61663 160 16.15885 12.05067 14.31017 42.2687649.5986 54.83421 −1.64609 −0.92272 −1.15473 800 51.0784 56.8298562.58131 59.86038 70.12216 85.41013 0.514403 1.845444 0.577367 400078.1924 77.16535 74.70045 69.49389 78.49913 77.452 4.526749 3.9215693.117783 20000 70.59226 82.30058 76.13831 66.56196 75.56719 78.708552.777778 3.921569 4.157044 100000 84.9709 85.99795 101.1982 74.5200782.68761 71.37871 2.469136 5.536332 3.00231

Finally, the rosette morphology that is evident upon synapse formationwas clearly observed only in the presence of the P-targeting agentantibody conjugate with C4-2 cells (FIG. 11).

Example 3: In Vivo PK and Antitumor Activity

We first evaluated the pharmacokinetics of the P-targeting agentantibody conjugate. Male Sprague Dawley rats (Charles River) wereinjected intravenously at time zero at 1 and 5 mg/kg for P-targetingagent antibody conjugate or 0.5 mg/kg for the unconjugated UCHT1 Fab.Blood was collected at regular intervals to 32 hours and processed tomeasure drug concentrations using a sandwich-ELISA. Interestingly, theP-targeting agent antibody conjugate showed significantly improvedcirculating half-life (˜5-6 hours) compared to the unconjugated Fab (˜1hour) perhaps due to the increased overall hydrophobicity of theDUPA-linker moiety after conjugation. Of note, P-targeting agentantibody conjugate has an improved serum half-life relative to smallbispecific scFvs such as BiTEs (Bispecific T cell Engager, ˜2 hourhalf-life in human) despite their similar molecular weight (˜50,000 Da).The enhanced serum half-life of P-targeting agent antibody conjugatetranslates into a human half-life of >1×/day dosing. Also, the smallsize of P-targeting agent antibody conjugate may be advantageous forpenetrating solid tumors.

We next established a mouse xenograft model to evaluate the in vivoefficacy of P-targeting agent antibody conjugate. ImmunodeficientNOD/SCID mice were subcutaneously injected with a mixture of 1×10⁶ C4-2cells and 2×10⁶ hPBMCs in matrigel (BD Bioscience). After 4 days,treatment was initiated by injecting 2 mg/kg of drug via the tail veinand continued for 10 days (n=6). In a control group, mice were injectedwith an unconjugated mixture of P-Phthal (3) and UCHT1 wild-type Fab,and another group of mice was injected with vehicle alone (n=6). Tumorgrowth was monitored by external caliper measurement. The mixture andvehicle group showed tumor outgrowth approximately two weeks afterimplantation. However, the treatment group did not develop any palpabletumors for up to 6 weeks (when all of the mice in the other two groupshad to be sacrificed due to the large sizes of the tumors) (FIG. 12,Table 3). Histology also confirmed the formation of solid tumors in micefrom the control groups (PBS and mixture group) while no solid tumorswere detected in the treatment group. After confirming the prophylacticefficacy of P-targeting agent antibody conjugate, we carried a xenograftmodel in which we delayed treatment until we observed the formation of apalpable tumor. In this treatment study, we used NOD/scid gamma (NSG,Jackson Laboratory) mice, which are known to be more suitable for immunereconstitution with human-derived cells. On day zero, 1×10⁶ C4-2 cellsin matrigel were subcutaneously injected and, after three days, 20×10⁶hPBMCs were separately injected into the peritoneal cavity. Thisseparate injection of hPBMCs further allows assessing the capability ofP-targeting agent antibody conjugate to redirect T cells form theperiphery to the site of tumor. Palpable solid tumors (˜150 mm³) wereformed in mice approximately two weeks after tumor implantation, atwhich time we started treatment via tail vein injection with 1 mg/kgP-targeting agent antibody conjugate for 10 days (n=7). On the first andfourth day of treatment, we co-injected 10×10⁶ of ex vivo expanded Tcells from the same donor via tail vein into all groups to supplementmore cytotoxic effector cells. After treatment was started, immediatetumor shrinkage was observed in the treatment group, whereas the vehiclegroup (n=7) again showed rapid tumor outgrowth.

TABLE 3 In -vivo efficacy studies of P-targeting agent antibodyconjugate Day Tumor Volume [mm³] P-targeting agent antibody conjugate 237.888 113.4 61.32 48.618 46.08 52.022 4 35.77 37.23 35.52 37.765 31.09836.72 7 31.968 21.78 44.376 60.04 43.2 43.8 11 29.127 42.282 43.09274.646 29.07 37.422 14 27.269 27.72 25.2405 38.766 29.106 29.106 1813.8355 36.686 20.8035 27.255 28.44 28.116 21 18.63 22.62 0 19.7945 020.475 25 18.009 26.18 23.79 22.32 11.088 10.962 28 14.413 40.95 21.16837.026 28.49 39.368 32 45.2965 74.493 0 24.36 28.86 14.64 36 36.682596.148 34.02 22.01 0 16.896 42 23.94 74.8 30.016 41.616 0 16.775Unconjugated mixture 2 46.512 52.65 65.934 51.714 51.205 67.068 4 37.18526.88 36.96 39.347 38.08 37.488 7 35.644 44.968 40.32 39.9 45.54 43.2 1154.32 37.422 54 43.12 44.968 34.125 14 41.208 30.66 51.264 49.217 55.4834.684 18 36.92 44.156 227.476 203.463 250.908 58.32 21 44.625 25.3125601.965 563.64 494.125 26.04 25 169.32 38.25 686.28 944.58 532.35 44.06428 180.5 63.882 757.68 1387.2 513.188 131.648 32 360.47 73.44 697.6375393.12 36 356.16 91.047 1130.96 766.233 42 519.294 57.646 PBS 2 40.05249.928 36.4 45.369 58.8 44.384 4 37.8 25.62 39.69 34.6385 62.123 25.56 732.319 36.652 33.744 43.911 67.716 51.128 11 27.702 39.36 40.664 63.12154.614 57.4275 14 35.6655 35.438 18.4 41.7075 37.296 30.03 18 143.64156.8 54.648 92.4 104.4 37.31 21 379.008 157.32 375.417 597.1875 297.5454.1875 25 427.68 121.968 701.22 702.1 810.16 48.048 28 842.996 209.0881163.986 1098.2 963.746 48.96 32 493.476 92.4 36 921.6 157.32 42 253.376

Example 4: Synthesis of Second Generation P-Linker Compounds

In this example, we have designed and synthesized a second generationP-linker compound, which showed significantly improved activity comparedto the first generation P-linkers in the PSMA enzyme inhibition assay.The in vitro and in vivo efficacy and PK of the second generationP-linker compound is evaluated as described above.

An orthotopic prostate cancer xenograft model in immunodeficient mice(NOD/SCID gamma) allows us monitor the migration and penetration ofadoptively transferred human T-cells into tumors in the prostate gland.An isograft prostate cancer model in immunocompetent mice to is alsoutilized to further examine the in vivo activity and safety of targetingagent antibody conjugate. For that purpose, a mouse prostate cancer cellline, RM-1-hPSMA, which is syngeneic in B6 mice is used. To recruitmouse T-cells, a mouse specific αCD3 Fab (2C11) is conjugated withP-linkers as described above. Importantly, mPSMA strongly binds DUPA(the parent enzyme inhibitor of P-linkers) which enables assessment ofefficacy and safety in mice. The pharmacokinetics and safety ofP-targeting agent antibody conjugate is evaluated in a non-humanprimate, cynomolgus macaque (cyno) model. In previous reports, glutamatecarboxy peptidase activity of cynoPSMA was efficiently inhibited bysubstrate analogue inhibitors (such as DUPA) and therefore should havehigh affinity to our DUPA-linker. A cynomolgus monkey/human crossreactive targeting agent antibody conjugate is prepared by theconjugation of DUPA-linker to a high affinity human/cynomolguscross-reactive αCD3 Fab (SP34). The goal of these studies is to providethe requisite information to guide a Phase I dose escalating study inhumans.

Example 5: Complete Structure-Activity Relationship Study to DetermineOptimal Targeting Agent Antibody Conjugate Candidate

We have found that the affinity of P-linker compounds significantlyaffects target cell binding of the resulting P-targeting agent antibodyconjugates, which in turn affect their cytotoxic activity in cell-basedassays. To further improve the affinity of P-linker, we re-examinedprevious structure-activity relationship (SAR) data and the co-crystalstructures of PSMA-inhibitor complexes. Based on the analysis, wedesigned a second generation P-linker candidate, P-TriA (FIG. 15JCompound 14), in which the C-9 amide group in P-Phthal is substitutedwith a triazole linkage, and a shorter linear hydrocarbon linker wasused to connect two hydrophobic aromatic groups, triazole andphthalimide groups (FIG. 13).

PSMA Inhibition Assay

A 10 mM solution of N-acetyl-aspartyl-glutamate (NAAG) in 40 mM NaOH wasdiluted to 40 μM in Reaction Buffer (0.1 M Tris-HCl, pH=7.5), and thesolution was added to a 384 well plate (10 μL per well). For K_(M)measurements and K_(M) negative controls (absence of PSMA), the NAAGsolution was serially diluted 2-fold to obtain final NAAG concentrationsranging 40 μL-312.5 nM. For IC₅₀ measurements, the targeting agent PSMAinhibitors in Reaction Buffer containing 40 NM NAAG solution wasserially diluted 5-fold to obtain final inhibitor concentrations ranging100 μL-51.2 pM. To initiate reactions, 10 μL of rhPSMA (20 M in ReactionBuffer, R&D research) was added to each well. Reaction Buffer (10 μL)was added to the K_(M) control series. The plate was incubated at 37° C.for 30 min, and then heated to 95° C. for 3 min. Levels of glutamic acidwere quantified using Amplex® Red Glutamic Acid/Glutamate Oxidase AssayKit (Invitrogen). Fluorescence intensities were measured using aSpectraMax Gemini EM microplate reader (GMI) with excitation andemission filters of 545 and 590 nm, respectively. Ki values werecalculated using the Cheng-Prusoff equation from IC₅₀ and K_(M) valuesand these values were calculated using GraphPad Prism software. K_(M)value (0.288 μM) is consistent with that reported in the literature(Humblet, V.; Misra, P.; Bhushan, K. R.; Nasr, K.; Ko, Y.-S.; Tsukamoto,T.; Pannier, N.; Frangioni, J. V.; Maison, W. J. Med. Chem. 2009, 52,544-550). All reported data represent the mean of triplicateexperiments.

FIG. 16A and Table 4 depict a representative PSMA inhibition curve bydifferent inhibitors (P-Pthal, P-DNP, and P-TriA). The known PSMAinhibitor PMPA was used as a control for the experiment.

TABLE 4 PSMA Inhibition Assay Relative Fluorescence Units [μM] PhthalDNP TriA PMPA 50 581.035 525.6755 559.8246667 549.6583333 10 688.1815581.8336667 599.969 567.78 2 798.8586667 586.843 562.8576667 599.21166670.4 1021.402333 622.7463333 586.721 651.3216667 0.08 1273.804667704.2426667 664.949 787.839 0.016 1387.603 896.7893333 817.13033331118.747667 0.0032 1542.606 1123.0625 950.487 1386.251333 0.000641633.913 1413.9875 1259.409667 1575.243667 0.000128 1592.54451558.430667 1499.6145 1633.926333 0.0000256 1608.653333 1650.5191618.877333 1622.5365

FIG. 16B and Table 5 depict Km curve obtained from the same experimentdescribed in FIG. 16A and Table 4.

TABLE 5 K_(M) measurement NAAG [μM] RFU 20 653.474 10 549.5883333 5480.622 2.5 446.9936667 1.25 434.8145 0.625 422.2223333 0.3125 400.64550.15625 403.0393333

As shown in Table 6, the enzyme-based inhibition assay revealed morethan 400-fold enhanced affinity for the P-TriA (average Ki=4.8 pM)compared to P-Phthal (average Ki=2 nM).

TABLE 6 Phthal IC₅₀ DNP IC₅₀ TriA IC₅₀ PMPA IC₅₀ Trial (Ki) (Ki) (Ki)(Ki) Km 1 2.08 × 10⁻⁷M 7.28 × 10⁻⁹M 1.62 × 10⁻¹⁰M 1.11 × 10⁻⁸M 2.88 ×10⁻⁷M (2.95 × 10⁻⁹M) (2.95 × 10⁻¹⁰M) (2.30 × 10⁻¹²M) (1.58 × 10⁻¹⁰M) 23.97 × 10⁻⁷M 4.46 × 10⁻⁹M 1.09 × 10⁻⁹M 2.48 × 10⁻⁸M 1.09 × 10⁻⁷M (2.15 ×10⁻⁹M) (2.42 × 10⁻¹¹M) (5.91 × 10⁻¹²M) (1.34 × 10⁻¹⁰M 3 2.23 × 10⁻⁷M4.58 × 10⁻⁹M 1.63 × 10⁻⁹M 1.66 × 10⁻⁸M 7.55 × 10⁻⁷M (8.39 × 10⁻¹⁰M)(1.72 × 10⁻¹¹M) (6.13 × 10⁻¹²M) (6.24 × 10⁻¹¹M) Average 1.98 × 10⁻⁹M4.82 × 10⁻¹¹M 4.78 × 10⁻¹²M 1.18 × 10⁻¹⁰M Ki

In Vitro Cytotoxicity Assays

Peripheral blood mononuclear cells (PBMCs) were purified from freshhealthy human donor blood by conventional Ficoll-Hypaque gradientcentrifugation. Purified PBMCs were washed and incubated in flasks inRPMI media with 10% FBS for 1 hours to remove any adherent cells. C4-2(PSMA+) cells (target cells) were dissociated with 0.05% tryspin/EDTAsolution (HyClone) and washed with RPMI with 10% FBS. 1×10⁴ target cellswere mixed with PBMCs at 1:10 ratio in 100 mL, and incubated withdifferent concentrations of conjugated and unconjugated Fabs (10 mL inPBS) for 24 and 48 hours at 37° C. Cytotoxicity of each well wasmeasured for LDH (lactate dehydrogenase) levels in supernatant usingCytotox-96 non-radioactive cytotoxicity assay kit (Promega). Lysissolution (10 mL, provided in the same kit) was added to wells with onlytarget cells to get the maximum killing, and spontaneous killing wasmeasured from wells with effector and target cells treated with vehicle(10 mL PBS). The absorbance at 490 nm was recorded using SpectraMax 250plate reader (Molecular Devices Corp.). Percent cytotoxicity wascalculated by:

%Cytotoxicity=(Absorbance_(expt)−Absorbance_(spontaneous average))/(Absorbance_(max killing average)−Absorbance_(spontaneous average))×100

FIG. 17A-B shows the cytoxicity at 24 hours and 48 hours, respectively.Table 7 shows the numerical values of the graphs depicted in FIG. 17A-B.

TABLE 7 In vitro cytotoxicity assay % Cytotoxicity double-Phthaldouble-TriA [pM] conjugate conjugate wt-UCHT1 24 h 0 0 0 0 0.0512−0.892388451 −1.319308255 −0.175284838 0.256 −0.699912511 −1.604564093−0.876424189 1.28 −0.699912511 −1.497593154 −0.911481157 6.4 −0.314960632.31770369 −0.473269062 32 −0.472440945 13.06828312 −0.753724803 16011.65354331 33.17881975 −0.403155127 800 31.14610674 36.147263331.191936897 4000 33.14085739 37.93902656 1.858019281 48 h 0 0 0 00.01024 −1.277711965 −1.636828645 −0.608963949 0.0512 −0.913860213−1.227621483 −1.023059435 0.256 −1.827720426 −1.585677749 −0.5196492371.28 −1.523100355 4.228473998 −0.081195193 6.4 1.455407006 13.04347826−0.487171159 32 16.68641056 37.92838875 −1.104254628 160 38.2636655942.01193521 −0.941864242 800 38.09443222 38.72122762 4.871711595

In another experiment, P-TriA is conjugate to αCD Fab to generate asecond generation P-targeting agent antibody conjugate. The in vitroactivity and in vivo PK and efficacy of the second generationP-targeting agent antibody conjugate is compared with the firstgeneration P-targeting agent antibody conjugate. The in vitro efficacyof the targeting agent antibody conjugate using other humanPSMA-positive prostate cancer cell lines including 22Rv1 and PC-3-huPSMAis also evaluated. In addition, the activity of the targeting agentantibody conjugate is also tested in patient-derived prostate cancertissue. The published tissue-specificity of the P-linker moiety using acorresponding fluorescein isothiocyanate (FITC) conjugate and frozentissue microarray (Asterand) may also be confirmed. In addition to thesubcutaneous xenograft model described above, the optimal P-targetingagent antibody conjugate construct in an orthotopic prostate cancermodel, which more closely mimics the specific tumor microenvironment ofthe prostate gland, may also be determined. In order to determine theoptimal P-targeting agent antibody conjugate construct, a luciferizedprostate cancer cell line (LNCaP-Luc) is used to monitor tumor growthand regression by bioluminescence imaging. On day 0, cancer cells(1.0×10⁶ cells) are injected in the posterior prostatic lobe in 6-weekold NSG mice. After two weeks, freshly purified hPBMCs areintraperitoneally injected. Tumor growth is monitored twice per week andincreases in tumor bioluminescence are expected to be observed around4-week post tumor implantation. Prior to starting treatment, mice arebled and correct immune reconstitution is assessed by detecting human Tcells in periphery using FACS. During treatment, 0.2-1 mg/kg ofP-targeting agent antibody conjugate is intravenously administered dailyfor up to 10 days. Tumor growth is monitored up to 8 weeks. IHC studiesare performed to detect infiltrated T-cells in the prostate gland.

Example 6: Safety and Efficacy Studies in Immunocompetent Mice

In order to accurately assess drugs and to optimize the treatmentprotocol, a mouse surrogate P-targeting agent antibody conjugate thatmay recognize mouse T cells is generated. A hamster monoclonal 145-2C11that binds mouse CD3 (this antibody also binds epsilon subunit as UCHT1does) is used in the generation of the mouse surrogate P-targeting agentantibody conjugate. The variable region sequence of 145-2C11 wasobtained from the literature, and the synthetic gene is cloned into thepBAD expression vector to express pAcF-containing Fab in E. coli. We areconfirming the binding of m-P-targeting agent antibody conjugate usingFACS and its in vitro activity is measured using purified mPBMCs. OurP-linker moieties are based on the hPSMA enzyme inhibitors, which haveshown similar activity against various PSMAs from different originsincluding mouse, rat, dog, and monkey. Therefore the optimizedDUPA-linker compound we developed in previous studies may be directlyconjugated to an anti-m-CD3 antibody to quickly generate the mousesurrogate version of P-targeting agent antibody conjugate (mP-targetingagent antibody conjugate). However, unlike human, the expression levelsof mPSMA in normal mouse prostate tissues are very low, andover-expression in known mouse prostate cancer cell lines iscontroversial. To mimic the human PSMA expression levels, therefore, weare using the mouse prostate cancer cell line RM-1 that is stablytransduced with human PSMA (RM-1-hPSMA). The parent RM-1 cell line isMHC 1-deficient and syngeneic in C57BL/6 (B6) mice and it is known thatRM-1-hPSMA also grows well in immunocompetent B6 mice. We may firstconfirm the in vitro activity of mP-targeting agent antibody conjugateagainst RM-1-hPSMA in the presence of mPBMCs. We may then measure thepharmacokinetic parameters, which together with the in vitro activityexperiments determine the dosing paradigm for the efficacy studies. Forin vivo studies, we are orthotopically injecting 0.5×10⁶ RM-1-hPSMAcells into B6 mice. At ˜day 10, when the tumors reach ˜200 mm³, mice aretreated by intravenously injecting mP-targeting agent antibodyconjugate. We determine the minimal efficacious dose, optimal dosingfrequency, and maximum tolerated dose. We also assess theimmuno-toxicity of mP-targeting agent antibody conjugat in B6 mice invarious organs (e.g., brain, liver, heart, kidneys, spleen, lung, etc.).Of note, although there has been no direct comparison, some reportssuggest that there are significant amounts of mPSMA expressions in mousekidney compared with human, which should be considered during theassessment of kidney toxicity. Body weight, elevated cytokine levels(TNF-α, IFN-γ, IL-6, IL-2 etc.), and activation status of T-cells (CD69and CD25-positive) are measured during the treatment to monitor thedose-related side effects such as cytokine release syndrome in mice. Webelieve the dose dependent efficacy data together with the maximumtolerated dose and pharmacokinetic parameters determined in this studyprovides guidance for a safe starting dose in the non-human primatestudy, as well as a better estimate of the efficacious dose in man.

Example 7. Generation of a Human/Cynomolgus Monkey Cross-ReactiveP-Targeting Agent Antibody Conjugate and Safety Studies in Non-HumanPrimates

We are evaluating the pharmacokinetics and safety of P-targeting agentantibody conjugate in a non-human primate, cynomolgus macaque (cyno)model. Unfortunately, we have determined that the UCHT1 is notsufficiently cyno cross-reactive to allow safety assessment in NHPs.Therefore a human/cynomolgus monkey cross-reactive P-targeting agentantibody conjugate (cyP-targeting agent antibody conjugate) is preparedby the conjugation of the P-linker to human/cynomolgus cross-reactiveαCD3 Fab (SP34), which also binds the CD3 epsilon chain with highaffinity (Kd=4 nM). As SP34 is originally derived from mouse, we aremaking a chimeric or humanized version of SP34. As discussed above, ourP-linker compound should have similar affinity with cyno-PSMAs. Weconfirm the binding with CHO cells transfected with cynoPSMA. In vitrocytotoxicity against C4-2 cells is measured in the presence of cyPBMCs.We also perform similar in vitro cytotoxicity assays using purifiedhuman and cyno T-cells, and compare their responses (magnitude of targetcell lysis, efficacy, T-cell activation status, and cytokine release).We also assess the efficacy of the bispecific in the rodent C4-2xenograft model and carry out PK, dose escalation and dose frequencystudies as described above.

For the in vivo NHP PK studies, male cynomolgus monkeys intravenouslyreceive vehicle control or cyP-targeting agent antibody conjugate. Bloodsamples be collected at regular time points to determine PK parameters.We are using the cyno PK data together with the rodent PK, safety andefficacy study data to design an ascending dose cyno safety study, wheregroups of three male cynomolgus monkey intravenously receive differentamounts of cyP-targeting agent antibody conjugate up to three weeks(with Charles River, SNBL, or Covance). During the safety study, animalsare observed for mortality or moribundity as well as clinical signs orreactions to treatment. Other analyses are conducted including bodytemperature, ophthalmology, body weight, food consumption,cardiovascular function, hematology, coagulation, serum chemistry, urineanalysis, lymphocyte subtyping (FACS), cytokine release, andtoxicokinetics. Serum is also prepared for measuring serum concentrationlevels of cyP-targeting agent antibody conjugate and for immunogenicityevaluation. After treatment, tissues and organs from animals aresubjected to a thorough macroscopic assessment as well ashistopathological examination. The goal of these studies and the rodentstudies is to provide the requisite data for a Phase I dose escalatingstudy in humans.

Example 8: Targeting Agent Antibody Conjugate Production Optimization,Stability Tests, and Development of Bioanalytical Methods

In this example, we are developing an optimized protocol for theproduction of P-targeting agent antibody conjugate to provide enoughmaterial for the studies disclosed herein. The humanized SP34 sequenceis sub-cloned into different types of fermentation vectors to screen foroptimal inducible systems. We also optimize E. coli cell strains as wellas other fermentation parameters and determine the stability of theexpression system. Antibodies from fermentation batches are comparedwith ones from previous small-batch expression to ensure their purityand activity. Targeting agent conjugation conditions (temperature, pH,concentrations, and ligand-to-antibody ratio) are optimized forlarge-scale reactions. Alternative purification methods are tested toreadily remove excess targeting agents after the large-scaleconjugation. We test the serum stability of P-targeting agent antibodyconjugate by incubation in pooled human serum at 37° C. under sterileconditions. Aliquots are analyzed at timed intervals for theconcentration of intact P-targeting agent antibody conjugate anddegradation products (e.g., free DUPA-linkers) by LS-MS. In vitrocytoxcicity is also performed to determine the concentrations offunctionally active P-targeting agent antibody conjugate in serum. Thethermal stability profile is assessed by differential scanningcalorimetry (DSC) to determine the T_(m) of the P-targeting agentantibody conjugate compared with the parent SP34. Long-term stability istested at 4° C. over at least 2 months at different concentrations (1-50mg/ml) in various pre-formulation buffers. We are also developingbioanalytical methods to more accurately determine pharmacokineticparameters of P-targeting agent antibody conjugate. In pharmacokineticstudies, to only detect functionally intact P-targeting agent antibodyconjugate s, huPSMA-coated ELISA plates are used to capture P-targetingagent antibody conjugate in serum followed by incubation with purifiedCD3 epsilon chain with hexahistidine tag (His-tag) (SEQ ID NO: 41).After incubation with secondary aHis-Tag antibodies conjugated withhorse radish peroxidase, signals are visualized using a chemiluminescentkit. For immunogenicity studies, P-targeting agent antibody conjugate iscovalently immobilized to a sensor chip, and serum antibody responsesagainst P-targeting agent antibody conjugate is analyzed by surfaceplasmon resonance (SPR) detection. The specificity of binding toP-targeting agent antibody conjugate observed in serum samples isconfirmed by competition experiments using free P-targeting agentantibody conjugate.

Example 9: Synthesis of(S)-2-(3-((S)-5-(4-(6-(4-(1-(aminooxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-ylcarbamoyl)benzamido)hexyl)-1H-1,2,3-triazol-1-yl)-1-carboxypentyl)ureido)pentanedioicacid (P-TriA) Synthesis of (10S,14S)-tri-tert-butyl2,2-dimethyl-4,12-dioxo-3-oxa-5,11,13-triazahexadecane-10,14,16-tricarboxylate

As shown in the reaction scheme depicted in FIG. 15A, to a solution of 1(5.83 g, 19.7 mmol) in CH₂Cl₂ (150 mL) was added triphosgene (2.92 g,9.85 mmol). The reaction mixture was cooled to −78° C. and slowly addedEt₃N (27.5 mL, 0.197 mol). After the reaction mixture was stirred for 1h at ambient temperature, a solution of 2 (Maindron, N.; Poupart, S.;Hamon, M.; Langlois, J.-B.; Ple, N.; Jean, L.; Romieu, A.; Renard, P.-Y.Org. Biomol. Chem., 2011, 9, 2357-2370) in CH₂Cl₂ (10 mL) added. Afterstirring for 14 h at ambient temperature, the reaction mixture wasquenched with saturated aq. NH₄Cl. The aqueous layer was extracted withCH₂Cl₂ and the combined organic layers were dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (EtOAc:n-hexane=1:3) to afford 11.2 g (97%)of 3 as a pale yellow oil: R 0.5 (EtOAc:n-hexane=1:2); ¹H NMR (400 MHz,CDCl₃) δ 5.50 (d, 1H, J=8.3 Hz), 5.46 (d, 1H, J=8.0 Hz), 5.01 (s, 1H),4.31 (m, 2H), 3.04 (m, 2H), 2.26 (m, 2H), 2.03 (m, 1H), 1.83 (m, 1H),1.70 (m, 1H), 1.58 (m, 1H), 1.41 (s, 9H), 1.40 (s, 9H), 1.39 (s, 9H),1.38 (s, 9H), 1.32 (m, 4H); LR-MS (ESI+) m/z 588 (M+H⁺).

Synthesis of (S)-di-tert-butyl2-(3-((S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl)ureido)pentanedioate

As shown in the reaction scheme depicted in FIG. 15B, to a solution of 3(10.8 g, 18.4 mmol) in CH₂Cl₂ (100 mL) were added 2,6-lutidine (4.29 mL,36.8 mmol) and TMSOTf (5.00 mL, 27.6 mmol) at 0° C. After stirring for30 min at ambient temperature, the reaction mixture was quenched withMeOH and concentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (EtOAc:n-hexane=3:1 to MeOH:EtOAc=1:10) toafford 8.09 g (90%) of 4 as an yellow oil: R_(f)0.4 (MeOH:EtOAc=1:10);LR-MS (ESI+) m/z 488 (M+H⁺).

Synthesis of (S)-di-tert-butyl2-(3-((S)-6-azido-1-tert-butoxy-1-oxohexan-2-yl)ureido)pentanedioate

As shown in the reaction scheme depicted in FIG. 15C, to a solution ofsodium azide (1.55 g, 23.8 mmol) in CH₃CN (30 mL) was added dropwiseTf₂O (3.20 mL, 19.0 mmol) at 0° C. After 1h, to a solution of 4 (5.80 g,11.9 mmol), Et₃N (4.98 mL, 35.7 mL) and CuSO₄ (38.0 mg, 0.238 mmol) inCH₃CN (mL) was added dropwise above the triflic azide solution at 0° C.After stirring for 14 h at ambient temperature, the reaction mixture wasquenched with saturated aq. NH₄Cl. The aqueous layer was extracted withEtOAc and the combined organic layers were dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (EtOAc:n-hexane=1:3) to afford 3.20 g (52%)of 5 as white solid: R_(f)0.3 (EtOAc:n-hexane=1:2); ¹H NMR (400 MHz,CDCl₃) δ 6.33 (s, 2H), 5.22 (m, 2H), 4.28 (m, 2H), 3.26 (t, 2H, J=6.8Hz), 2.28 (m, 2H), 2.02 (m, 1H), 1.86 (m, 1H), 1.77 (m, 1H), 1.61 (m,3H), 1.46 (s, 18H), 1.43 (s, 9H); LR-MS (ESI+) m/z 514 (M+H⁺).

Synthesis of tert-butyl oct-7-ynylcarbamate

As shown in the reaction scheme depicted in FIG. 15D, to a solution of 6(Coutrot, F.; Romuald, C.; Busseron, E. Org. Lett., 2008, 10, 3741-3744)(101 mg, 0.807 mmol) in THF/H₂O (4/4 mL) were added NaHCO₃ (102 mg, 1.21mmol) and Boc₂O (0.210 mL, 0.968 mmol). After stirring for 14 h atambient temperature, the reaction mixture was quenched with saturatedaq. NH₄Cl. The aqueous layer was extracted with EtOAc and the combinedorganic layers were dried over MgSO₄ and concentrated in vacuo. Theresidue was purified by flash column chromatography on silica gel(EtOAc:n-hexane=1:7) to afford 144 mg (79%) of 7 as a pale yellow oil:R_(f)0.5 (EtOAc:n-hexane=1:7); LR-MS (ESI+) m/z 226 (M+H⁺).

Synthesis of (S)-di-tert-butyl2-(3-((S)-1-tert-butoxy-6-(4-(6-(tert-butoxycarbonylamino)hexyl)-1H-1,2,3-triazol-1-yl)-1-oxohexan-2-yl)ureido)pentanedioate

As shown in the reaction scheme depicted in FIG. 15E, to a solution of 5(770 mg, 1.25 mmol), 7 (309 mg, 1.37 mmol) and sodium ascorbate (99.1mg, 0.500 mmol) in THF (20 mL) was added a solution of CuSO₄*H₂O (62.4mg, 0.250 mmol) in H₂O (3.2 mL). After stirring for 30 min at ambienttemperature, the aqueous layer was extracted with EtOAc and the combinedorganic layers were dried over MgSO₄ and concentrated in vacuo. Theresidue was purified by flash column chromatography on silica gel(EtOAc:n-hexane=2:1) to afford 644 mg (70%) of 8 as white solid:R_(f)0.3 (EtOAc:n-hexane=2:1); ¹H NMR (400 MHz, CDCl₃) δ 5.28 (m, 2H),4.57 (s, 1H), 4.30 (m, 4H), 3.08 (m, 2H), 2.67 (t, 2H, J=7.7 Hz), 2.30(m, 2H), 2.04 (m, 2H), 1.89 (m, 2H), 1.78 (m, 2H), 1.64 (m, 3H), 1.44(s, 9H), 1.41 (s, 27H), 1.37 (m, 8H); LR-MS (ESI+) m/z 739 (M+H⁺).

Synthesis of (S)-di-tert-butyl2-(3-((S)-6-(4-(6-aminohexyl)-1H-1,2,3-triazol-1-yl)-1-tert-butoxy-1-oxohexan-2-yl)ureido)pentanedioate

As shown in the reaction scheme depicted in FIG. 15F, to a solution of 8(644 mg, 0.872 mmol) in CH₂Cl₂ (10 mL) were added 2,6-lutidine (0.200mL, 1.74 mmol) and TMSOTf (0.240 mL, 1.31 mmol) at 0° C. After stirringfor 30 min at ambient temperature, the reaction mixture was quenchedwith MeOH and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel (EtOAc:n-hexane=3:1 toMeOH:EtOAc=1:10) to afford 351 mg (63%) of 9 as an yellow oil: R_(f)0.2(MeOH:EtOAc=1:10); LR-MS (ESI+) m/z 639 (M+H⁺).

Synthesis of methyl4-(2,2-dimethyl-4,8-dioxo-3,6,12,15,18-pentaoxa-5,9-diazaicosan-20-ylcarbamoyl)benzoate

As shown in the reaction scheme depicted in FIG. 15G, to a solution of10 (Hagemeyer, C; Peter, K.; Johnston, A. P. R.; Owen, D. PCT Int. Appl.WO 2012142659 A1, 2012) (74.5 mg, 0.209 mmol) in CH₂Cl₂ (5 mL) wereadded i-PrNEt (54.7 μL, 0.314 mmol) and 4-(chlorocarbonyl)benzoic acidmethyl ester (125 mg, 0.627 mmol) at 0° C. After stirring for 12 h atambient temperature, the reaction mixture was quenched with saturatedaq. NH₄Cl. The aqueous layer was extracted with CH₂Cl₂ and the combinedorganic layers were dried over MgSO₄ and concentrated in vacuo. Theresidue was purified by flash column chromatography on silica gel(MeOH:EtOAc=1:20) to afford 98.1 mg (89%) of 11 as a pale yellow oil:R_(f)0.4 (MeOH:EtOAc=1:10); LR-MS (ESI+) m/z 550 (M+Na⁺).

Synthesis of4-(2,2-dimethyl-4,8-dioxo-3,6,12,15,18-pentaoxa-5,9-diazaicosan-20-ylcarbamoyl)benzoicacid

As shown in the reaction scheme depicted in FIG. 15H, to a solution of11 (200 mg, 0.379 mmol) in THF/H₂O (5/5 mL) was added LiOH.H₂O (23.9 mg,0.569 mmol). After stirring for 4 h at ambient temperature, the reactionmixture was acidified with 2N HCl. The aqueous layer was extracted withEtOAc and the combined organic layers were dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (MeOH:EtOAc=1:7) to afford 118 mg (74%) of12 as a pale yellow oil: R_(f)0.2 (MeOH:EtOAc=1:7); LR-MS (ESI+) m/z 536(M+Na⁺).

Synthesis of (S)-di-tert-butyl2-(3-((S)-1-tert-butoxy-6-(4-(6-(4-(2,2-dimethyl-4,8-dioxo-3,6,12,15,18-pentaoxa-5,9-diazaicosan-20-ylcarbamoyl)benzamido)hexyl)-1H-1,2,3-triazol-1-yl)-1-oxohexan-2-yl)ureido)pentanedioate

As shown in the reaction scheme depicted in FIG. 15I, to a solution of 9(45.8 mg, 71.7 μmol) and 12 (43.8 mg, 71.7 μmol) in DMF (5 mL) wereadded EDCI (41.2 mg, 0.215 mmol), HOBt (29.1 mg, 0.215 mmol) and Et₃N(30.0 μL, 0.215 mmol) at 0° C. After stirring for 14 h at ambienttemperature, the reaction mixture was quenched with saturated aq. NH₄Cl.The aqueous layer was extracted with EtOAc and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel (MeOH:EtOAc=1:15)to afford 22.0 mg (27%) of 13 as a pale yellow oil: LR-MS (ESI+) m/z1134 (M+H⁺).

Synthesis of(S)-2-(3-((S)-5-(4-(6-(4-(1-(aminooxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-ylcarbamoyl)benzamido)hexyl)-1H-1,2,3-triazol-1-yl)-1-carboxypentyl)ureido)pentanedioicacid (P-TriA)

As shown in the reaction scheme depicted in FIG. 15J, to a solution of13 (26.6 mg, 23.4 μmol) in CH₂Cl₂ (1.5 mL) was added TFA (1.5 mL). Afterstirring for 14 h at ambient temperature, the reaction mixture wasconcentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (MeOH:EtOAc=1:15) to afford 20.1 mg (99%)of 14 as a pale yellow oil: LR-MS (ESI+) m/z 866 (M+H⁺). The ESI-MSanalysis and deconvoluted mass spectrum of P-TriaA (compound 14)conjugated to an anti-CD3 Fab (UCHT1) pAcF mutant (LC-202X/HC-138X) aredepicted in FIGS. 15K and 15L, respectively.

TABLE 8Sequences of antibodies, antibody fragments or targeting agents (sitesfor UCHT1 unnatural amino acid incorporation are underlined) DescriptionSEQUENCE  1 Light chain of ATGAAAAAGAATATCGCATTTCTTCTTGCTAGC an anti-CD3ATGTTCGTTTTTTCTATTGCTACAAACGCATAC clone ofGCTGACATCCAGATGACCCAGTCTCCATCCTCC UCHT1 CTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATCCGTAATT ATCTGAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTATACCTCCCGC CTGGAGTCTGGGGTCCCATCAAGGTTCAGTGGCTCTGGATCTGGGACAGATTACACTCTGACCAT CAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGGTAATACTCTGCCGTGG ACGTTCGGCCAAGGTACCAAGGTGGAGATCAA ACGAACT GTGGCTGCACCATCTGTCTTCATCTT CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCT ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAG CAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG TCC TCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T  2Hevy chain of ATGAAAAAGAATATCGCATTTCTTCTTGCATCT an anti-CD3ATGTTCGTTTTTTCTATTGCTACAAACGCGTAC clone ofGCTGAGGTGCAGCTGGTGGAGTCTGGAGGAGG UCHT1 CTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTACTCCTTTACCGGCTA CACTATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCACTGATTAATCCT TATAAAGGTGTTTCCACCTATAACCAGAAATTCAAGGATCGATTCACCATCTCCGTAGATAAATC CAAAAACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT GCTAGAAGCGGATACTACGGCGATAGTGACTGGTATTTTGACGTCTGGGGCCAAGGAACCCTGG TCACCGTCTCCTCA GCC TCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC AAG AGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTG TGACAAAACTCACACA  3 SS-14Ala-Gly-cyclo(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr- (somatostatinPhe-Thr-Ser-Cys) analog)  4 OC D-Phe1-cyclo(Cys2-Phe3-D-Trp4-Lys5-Thr6-(somatostatin Cys7)Thr(ol)8 analog)  5 TOCD-Phe1-cyclo(Cys2-Tyr3-D-Trp4-Lys5-Thr6- (somatostatin Cys7)Thr(ol)8analog)  6 TATE D-Phe1-cyclo(Cys2-Tyr3-D-Trp4-Lys5-Thr6- (somatostatinCys7)Thr8 analog)  7 NOC D-Phe1-cyclo(Cys2-1-NaI3-D-Trp4-Lys5-Thr6-(somatostatin Cys7)Thr(ol)8 analog)  8 NOC-ATED-Phe1-cyclo(Cys2-1-NaI3-D-Trp4-Lys5-Thr6- (somatostatin Cys7)Thr8analog)  9 BOC D-Phe1-cyclo(Cys2-BzThi3-D-Trp4-Lys5-Thr6- (somatostatinCys7)Thr(ol)8 analog) 10 BOC-ATED-Phe1-cyclo(Cys2-BzThi3-D-Trp4-Lys5-Thr6- (somatostatin Cys7)Thr8analog) 11 KE108 Tyr-cyclo(DAB-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe)(somatostatin analog) 12 LM3 p-Cl-Phe-cyclo(D-Cys-Tyr-D-Aph(Cbm)-LysThr-(somatostatin Cys)D-Tyr-NH2 analog) 13 BN(bombesinpGlu1-G1n2-Arg3-Leu4-Gly5-Asn6-Gln7-Trp8-Ala9- analog)Val10-Gly11-His12-Leu13-Met14-NH2 14 RP527N35-Gly-5-Ava-[Gln7-Trp8-Ala9-Val10-Gly11-His12- (bombesinLeu13-Met14-NH2] analog) 15 Demobesin 1N40-1-bzlg0[D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11- (bombesinHis12-Leu-NHEt13] analog) 16 Demobesin 4N4-[Pro1-Gln2-Arg3-Tyr4-Gly5-Asn6-Gln7-Trp8- (bombesinAla9-Val10-Gly11-His12-Leu13-Nle14-NH2] analog) 17 BBS-38(NαHis)Ac-β-Ala-β-Ala-[Gln7-Trp8-Ala9-Val10- (bombesinGly11-His12-Cha13-Nle14-NH2] analog) 18 BAY 86-43673-cyano-4-trimethylammonium-benzoyl-Ala(SO3H)- (bombesinAla(SO3H)-Ava-[Gln7-Trp8-Ala9-Val10-NMeGly11- analog)His12-Sta13-Leu14-NH2] 19 MGLeu1-Glu2-Glu3-Glu4-Glu5-Glu6-Ala7-Tyr8-Gly9- (minigastrinTrp10-Met11-Asp12-Phe13-NH2 analog) 20 MG0D-Glu1-Glu2-Glu3-Glu4-Glu5-Glu6-A1a7-Tyr8-Gly9- (minigastrinTrp10-Met11-Asp12-Phe13-NH2 analog) 21 MG11D-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (minigastrin analog) 22 H2-MetHis-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (minigastrin analog) 23H2-Nle His-His-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (minigastrin analog)24 Demogastrin N4-D-Glu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2(minigastrin analog) 25 Cyclo-MG1c(γ-D-Glu-Ala-Tyr-D-Lys)-Trp-Met-Asp-Phe-NH2 (minigastrin analog) 26MGD5 Gly-Ser-Cys(succinimidopropionyl-Glu-Ala-Tyr-Gly- (minigastrinTrp-Nle-Asp-Phe-NH2)-Glu-Ala-Tyr-Gly-Trp-Nle- analog) Asp-Phe-NH2 27Buserelin pGlu1-His2-Trp3-Ser4-Tyr5-D-Ser(tBu)6-Leu7-Arg8- (GnRH analog)Pro9-NHC2H5 28 GoserelinpGlu1-His2-Trp3-Ser4-Tyr5-D-Ser(tBu)6-Leu7-Arg8- (GnRH analog)Pro9-AzGly10-NH2 29 LeuprolidepGlu1-His2-Trp3-Ser4-Tyr5-D-Leu6-Leu7-Arg8-Pro9- (GnRH analog) NHC2H5 30Nafarelin pGlu1-His2-Trp3-Ser4-Tyr5-D-Nal(2)6-Leu7-Arg8- (GnRH analog)Pro9-NHC2H5 31 TriptorelinpGlu1-His2-Trp3-Ser4-Tyr5-D-Trp6-Leu7-Arg8-Pro9- (GnRH analog) Gly10-NH232 Abarelix Ac-D-Ala1-D-Cpa2-D-Ala3-Ser4-Tyr5-D-Asp6-Leu7- (GnRH analog)Ilys8-Pro9-D-Ala10-NH2 33 AcylineAc-D-Nal1-D-Cpa2-D-Pal3-Ser4-Aph(Ac)5-D- (GnRH analog)Aph(Ac)6-Leu7-Ilys8-Pro9-D-Ala10-NH2 34 AntarelixAc-D-Nal1-D-Cpa2-D-Pal3-Ser4-Tyr5-D-Hci6-Leu7- (GnRH analog)Ilys8-Pro9-D-Alal0-NH2 35 Antide (GnRHAc-D-Nal1-D-Cpa2-D-Pal3-Ser4-Lys(Nic)5-D- analog)Lys(Nic)6-Leu7-Ilys8-Pro9-D-Ala10-NH2 36 Azaline BAc-D-Nal1-D-Cpa2-D-Pal3-Ser4-Aph(Atz)5-D- (GnRH analog)Aph(Atz)6-Leu7-Ilys8-Pro9-D-Ala10-NH2 37 CetrorelixAc-D-Nal1-D-Cpa2-D-Pal3-Ser4-Tyr5-D-Cit6-Leu7- (GnRH analog)Arg8-Pro9-D-Ala10-NH2 38 Degarelix Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-Aph(L-(GnRH analog) hydroorotyl)5-D-Aph(carbamoyl)6-Leu7-Ilys8-Pro9-D-Ala10-NH2 39 Ganirelix Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-Tyr5-D-hArg(Et2)6-(GnRH analog) Leu7-hArg(Et2)8-Pro9-D-Ala10-NH2 40 OzarelixAc-D-Nal1-D-Cpa2-D-Pal3-Ser4-N-MeTyr5-D-hCit6- (GnRH analog)Nle7-Arg8-Pro9-D-Ala10-NH2

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

That which is claimed is:
 1. A compound of Formula XII:Y-L-A¹-L¹-A²-L²-A³-L³-X²  (Formula XII) wherein: Y is a ligand ofprostate specific membrane antigen (PSMA); L is

A¹ is selected from the group consisting of an aryl, a 5- to 6-memberedheteroaryl, —C(O)—, —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)N(R¹)—,—S(O)₂N(R¹)—, —N(R¹)S(O)— and —N(R¹)S(O)₂—; L¹ is

A² is selected from the group consisting of a bond, —C(O)—, —N(R¹)—,—O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)N(R¹)—, —S(O)₂N(R¹)—, —N(R¹)S(O)—and —N(R¹)S(O)₂—; L² is

A³ is a bond,

L³ is

X² is a linker bound to a functional group that reacts with an unnaturalamino acid, or a linker bound to a modified unnatural amino acid,wherein the modified unnatural amino acid is part of X, wherein X is amodified therapeutic peptide, protein, or antibody; each R¹ isindependently selected from H, alkyl, or haloalkyl; each R², R²¹, R²²,and R²³ is independently selected from H, halo, —OR¹, —CN, —SR¹, alkyl,cycloalkyl, haloalkyl, arylalkyl, or heteroarylalkyl; each R³ isindependently selected from halo, —OR¹, —CN, —SR¹, alkyl, cycloalkyl,haloalkyl, arylalkyl, or heteroarylalkyl, —NO₂, and NR¹R¹; each G¹ andG² is independently selected from the group consisting of a bond,—C(O)—, —N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)N(R¹)—,—S(O)₂N(R¹)—, —N(R¹)S(O)— and —N(R¹)S(O)₂—; each Z, Z¹, Z², and Z³ isindependently selected from the group consisting of a bond, —O—, and—N(R¹)—; k, k¹, k² and k³ are each independently selected from 0, 1, 2,3, 4, 5, 6, 7, 8, 9, and 10; m¹, m² and m³ are each independentlyselected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and p is 0, 1, 2, 3or 4, or a stereoisomer thereof.
 2. The compound of claim 1, wherein thecompound is of Formula XIIa:

wherein: Q is selected from the group consisting of:

 and E is selected from the group consisting of:


3. The compound of claim 2, wherein the compound is of Formula XIIb:


4. The compound of claim 3, wherein the compound is of Formula XIIc:


5. The compound of claim 2, wherein the compound is of Formula XIId:


6. The compound of claim 5, wherein the compound is of Formula XIIe:


7. The compound of claim 6, wherein the compound is of Formula XIIf:


8. The compound of claim 1, wherein: A¹ is —C(O)N(H)—.
 9. The compoundof claim 1, wherein: A¹ is


10. The compound of claim 1, wherein: A³ is


11. The compound of claim 10, wherein: A³ is


12. The compound of claim 1, wherein: each R², R²¹, R²², R²³, and R²⁴ isindependently selected from H, F, —CH₃, or —CF₃.
 13. The compound ofclaim 1, wherein: each R², R²¹, R²², R²³, and R²⁴ is H.
 14. The compoundof claim 1, wherein: X² is

wherein: A⁴ is selected from the group consisting of a bond, —C(O)—,—N(R′)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)N(R¹)—, —S(O)₂N(R¹)—,—N(R¹)S(O)— and —N(R¹)S(O)₂—; each R²⁴ is independently selected from H,halo, —OR¹, —CN, —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl, orheteroarylalkyl; k⁴ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and10; Z⁴ is selected from a bond, aryl, and a 5- to 6-membered heteroaryl;and X¹ is a functional group that reacts with an unnatural amino acidselected from: —ONH₂,

 —N₃,

 —N(H)NH₂, or —SH.
 15. The compound of claim 14, wherein: X² is


16. The compound of claim 15, wherein:


17. The compound of claim 1, wherein the compound is selected from:

and ONH₂ or a stereoisomer thereof.
 18. The compound of claim 1,wherein: X² is

wherein: A⁴ is selected from the group consisting of a bond, —C(O)—,—N(R¹)—, —O—, —C(O)N(R¹)—, —N(R¹)C(O)—, —S(O)N(R¹)—, —S(O)₂N(R¹)—,—N(R¹)S(O)— and —N(R¹)S(O)₂—; each R²⁴ is independently selected from H,halo, —OR¹, —CN, —SR¹, alkyl, cycloalkyl, haloalkyl, arylalkyl, orheteroarylalkyl; k⁴ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and10; Z⁴ is selected from a bond, aryl, and a 5- to 6-membered heteroaryl;and X³ is

 or —S—; X is a modified therapeutic peptide, protein, or antibody; L⁴is a bond directly attached to the modified unnatural amino acid, or alinker bound to the modified unnatural amino acid, wherein the modifiedunnatural amino acid is part of X.
 19. A composition comprising acompound of claim 1, wherein the purity of the compound is at least 90%.20. A method for treating a prostate cancer in a subject in needthereof, comprising administering a compound of claim 1.